32142, 21481, 25964, 21686, novel dehydrogenase molecules and uses therefor

ABSTRACT

The invention provides isolated nucleic acids molecules, designated DHDR nucleic acid molecules, which encode novel DHDR-related dehydrogenase molecules. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing DHDR nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a DHDR gene has been introduced or disrupted. The invention still further provides isolated DHDR proteins, fusion proteins, antigenic peptides and anti-DHDR antibodies. Diagnostic methods utilizing compositions of the invention are also provided.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/816,760, filed Mar. 23, 2001, which is acontinuation-in-part of U.S. patent application Ser. No. 09/634,955,filed Aug. 8, 2000, which claims the benefit of U.S. ProvisionalApplication Serial No. 60/192,002, filed Mar. 24, 2000. The entirecontents of all of the above-referenced applications are incorporatedherein by this reference.

BACKGROUND OF THE INVENTION

[0002] The oxidation and reduction of molecules is of criticalimportance in most metabolic and catabolic pathways in cells. A largefamily of enzymes which facilitate these molecular alterations, termeddehydrogenases, have been identified. In the forward reaction, theseenzymes catalyze the transfer of a hydride ion from the target substrateto the enzyme or a cofactor of the enzyme (e.g., NAD⁺ or NADP⁺), therebyforming a carbonyl group on the substrate. These enzymes are also ableto participate in the reverse reaction, wherein a carbonyl group on thetarget molecule is reduced by the transfer of a hydride group from theenzyme. Members of the dehydrogenase family are found in nearly allorganisms, from microbes to Drosophila to humans. Both between speciesand within the same species, dehydrogenases vary widely, and structuralsimilarities between distant dehydrogenase family members are mostfrequently found in the cofactor binding site of the enzyme. Even withina particular subclass of dehydrogenase molecules, e.g., the short-chaindehydrogenase molecules, members typically display only 15-30% aminoacid sequence identity, and this is limited to the cofactor binding siteand the catalytic site (Jornvall et al. (1995) Biochemistry34:6003-6013).

[0003] Different classes of dehydrogenases are specific for an array ofbiological and chemical substrates. For example, there existdehydrogenases specific for alcohols, for aldehydes, for steroids, andfor lipids, with particularly important classes of dehydrogenasesincluding the short-chain dehydrogenase/reductases, the medium-chaindehydrogenases, the aldehyde dehydrogenases, the alcohol dehydrogenases,and the steroid dehydrogenases. Within each of these classes, eachenzyme is specific for a particular substrate (e.g., ethanol orisopropanol, but not both with equivalent affinity). This exquisitespecificity not only permits tight regulation of the metabolic andcatabolic pathways in which these enzymes participate, without affectingsimilar but separate biochemical pathways in the same cell or tissue.The short-chain dehydrogenases, part of the alcohol oxidoreductasesuperfamily (Reid et al. (1994) Crit. Rev. Microbiol. 20:13-56), areZn⁺⁺-independent enzymes with an N-terminal cofactor binding site and aC-terminal catalytic domain (Persson et al. (1995) Adv. Exp. Med. Biol.372:383-395; Jornvall et al.(1995) supra), whereas the medium chaindehydrogenases are Zn⁺⁺-dependent enzymes with an N-terminal catalyticdomain and a C-terminal coenzyme binding domain (Jornvall et al.(1995)supra; Jornvall et al. (1999) FEBS Lett. 445:261-264). The steroiddehydrogenases are a subclass of the short-chain dehydrogenases, and areknown to be involved in a variety of biochemical pathways, affectingmammalian reproduction, hypertension, neoplasia, and digestion (Duax etal. (2000) Vitamins and Hormones 58:121-148). Aldehyde dehydrogenasesshow heterogeneity in the placement of these domains, and alsoheterogeneity in their substrates, which include toxic substances,retinoic acid, betaine, biogenic amine, and neurotransmitters (Hsu etal. (1997) Gene 189:89-94). It is common in higher organisms fordifferent dehydrogenase molecules to be expressed in different tissues,according to the localization of the substrate for which the enzyme isspecific. For example, different mammalian aldehyde dehydrogenases arelocalized to different tissues, e.g., salivary gland, stomach, andkidney (Hsu et al. (1997) supra).

[0004] Dehydrogenases play important roles in the production andbreakdown of nearly all major metabolic intermediates, including aminoacids, vitamins, energy molecules (e.g., glucose, sucrose, and theirbreakdown products), signal molecules (e g., transcription factors andneurotransmitters), and nucleic acids. As such, their activitycontributes to the ability of the cell to grow and differentiate, toproliferate, and to communicate and interact with other cells.Dehydrogenases also are important in the detoxification of compounds towhich the organism is exposed, such as alcohols, toxins, carcinogens,and mutagens.

[0005] A dehydrogenase of the short-chain family, 11-beta-hydroxysteroiddehydrogenase, activates glucocorticoids in the liver. Glucocorticoidsare known to induce transcription of hepatitis B virus (HBV) genes,probably by direct binding of the ligand-glucocorticoid receptor complexto an enhancer element in the HBV genome. There is also evidence thatshort chain dehydrogenases are transcriptional cofactors for retrovirusgene activation.

SUMMARY OF THE INVENTION

[0006] The present invention is based, at least in part, on thediscovery of novel members of the family of dehydrogenase molecules,referred to herein as DHDR nucleic acid and protein molecules (e.g.,DHDR-1, DHDR-2, DHDR-3, and DHDR-4). The DHDR nucleic acid and proteinmolecules of the present invention are useful as modulating agents inregulating a variety of cellular processes, e.g., viral infection,cellular proliferation, growth, differentiation, and/or migration.Accordingly, in one aspect, this invention provides isolated nucleicacid molecules encoding DHDR proteins or biologically active portionsthereof, as well as nucleic acid fragments suitable as primers orhybridization probes for the detection of DHDR-encoding nucleic acids.

[0007] In one embodiment, a DHDR nucleic acid molecule of the inventionis at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 89%, 90%, 95%,96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,99.8%, 99.9%, 99.99% or more identical to the nucleotide sequence (e.g.,to the entire length of the nucleotide sequence) shown in SEQ ID NO:1,3, 4, 6, 7, 9, 10, 12, 14, or 16, or the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC as Accession Number ______,PTA-1845, ______, ______, or ______ , or a complement thereof.

[0008] In a preferred embodiment, the isolated nucleic acid moleculeincludes the nucleotide sequence shown in SEQ ID NO:1, 3, 4, 6, 7, 9,10, 12, 14, or 16, or a complement thereof. In another embodiment, thenucleic acid molecule includes SEQ ID NO:3 and nucleotides 1-62 of SEQID NO:1. In another embodiment, the nucleic acid molecule includes SEQID NO:6 and nucleotides 1-330 of SEQ ID NO:4. In yet another embodiment,the nucleic acid molecule includes SEQ ID NO:9 and nucleotides 1-280 ofSEQ ID NO:7. In another embodiment, the nucleic acid molecule includesSEQ ID NO:12 and nucleotides 1-60 of SEQ ID NO:10. In anotherembodiment, the nucleic acid molecule includes SEQ ID NO:16 andnucleotides 1-101 of SEQ ID NO:14. In yet a further embodiment, thenucleic acid molecule includes SEQ ID NO:3 and nucleotides 2469-2660 ofSEQ ID NO:1. In another embodiment, the nucleic acid molecule includesSEQ ID NO:6 and nucleotides 1264-1379 of SEQ ID NO:4. In anotherembodiment, the nucleic acid molecule includes SEQ ID NO:9 andnucleotides 1388-1725 of SEQ ID NO:7. In another embodiment, the nucleicacid molecule includes SEQ ID NO:12 and nucleotides 1027-1209 of SEQ IDNO:10. In still another embodiment, the nucleic acid molecule includesSEQ ID NO:16 and nucleotides 1035-1108 of SEQ ID NO: 14. In anotherpreferred embodiment, the nucleic acid molecule consists of thenucleotide sequence shown in SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or16.

[0009] In another embodiment, a DHDR nucleic acid molecule includes anucleotide sequence encoding a protein having an amino acid sequencesufficiently identical to the amino acid sequence of SEQ ID NO:2, 5, 8,11, or 15, or an amino acid sequence encoded by the DNA insert of theplasmid deposited with ATCC as Accession Number ______, PTA-1845,______, ______, or ______. In a preferred embodiment, a DHDR nucleicacid molecule includes a nucleotide sequence encoding a protein havingan amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 92%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%,99.6%, 99.7%, 99.8%, 99.9%, 99.99% or more identical to the entirelength of the amino acid sequence of SEQ ID NO:2, 5, 8, 11, or 15, orthe amino acid sequence encoded by the DNA insert of the plasmiddeposited with ATCC as Accession Number ______, PTA-1845, ______,______, ______, or ______.

[0010] In another preferred embodiment, an isolated nucleic acidmolecule encodes the amino acid sequence of human DHDR-1, DHDR-2,DHDR-3, or DHDR-4. In another preferred embodiment, an isolated nucleicacid molecule encodes the amino acid sequence of mouse DHDR-2. In yetanother preferred embodiment, the nucleic acid molecule includes anucleotide sequence encoding a protein having the amino acid sequence ofSEQ ID NO:2, 5, 8, 11, or 15, or the amino acid sequence encoded by theDNA insert of the plasmid deposited with ATCC as Accession Number______, PTA-1845, ______, ______, or ______. In yet another preferredembodiment, the nucleic acid molecule is at least 50, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500,1550, 1600, 1650 1700, 1750, 1800, 1850, 1900, 1950, 2000 or morenucleotides in length. In a further preferred embodiment, the nucleicacid molecule is at least 50, 100, 150, 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700,1750, 1800, 1850, 1900, 1950, 2000 or more nucleotides in length andencodes a protein having a DHDR activity (as described herein).

[0011] Another embodiment of the invention features nucleic acidmolecules, preferably DHDR nucleic acid molecules, which specificallydetect DHDR nucleic acid molecules relative to nucleic acid moleculesencoding non-DHDR proteins. For example, in one embodiment, such anucleic acid molecule is at least 20, 30, 40, 50, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550,1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000 or more nucleotidesin length and hybridizes under stringent conditions to a nucleic acidmolecule comprising the nucleotide sequence shown in SEQ ID NO:1, 4, 7,10, or 14, the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number ______, PTA-1845, ______ ,______, or ______, or a complement thereof.

[0012] In preferred embodiments, the nucleic acid molecules are at least15 (e.g., 15 contiguous) nucleotides in length and hybridize understringent conditions to the nucleotide molecules set forth in SEQ IDNO:1, 4, 7, 10, or 14.

[0013] In other preferred embodiments, the nucleic acid molecule encodesa naturally occurring allelic variant of a polypeptide comprising theamino acid sequence of SEQ ID NO:2, 5, 8, 11, or 15, or an amino acidsequence encoded by the DNA insert of the plasmid deposited with ATCC asAccession Number ______, PTA-1845, ______, ______, or ______, whereinthe nucleic acid molecule hybridizes to a complement of a nucleic acidmolecule comprising SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16 understringent conditions.

[0014] Another embodiment of the invention provides an isolated nucleicacid molecule which is antisense to a DHDR nucleic acid molecule, e.g.,the coding strand of a DHDR nucleic acid molecule.

[0015] Another aspect of the invention provides a vector comprising aDHDR nucleic acid molecule. In certain embodiments, the vector is arecombinant expression vector. In another embodiment, the inventionprovides a host cell containing a vector of the invention. In yetanother embodiment, the invention provides a host cell containing anucleic acid molecule of the invention. The invention also provides amethod for producing a protein, preferably a DHDR protein, by culturingin a suitable medium, a host cell, e.g., a mammalian host cell such as anon-human mammalian cell, of the invention containing a recombinantexpression vector, such that the protein is produced.

[0016] Another aspect of this invention features isolated or recombinantDHDR proteins and polypeptides. In one embodiment, an isolated DHDRprotein includes at least one or more of the following domains: atransmembrane domain, a signal peptide domain, an aldehyde dehydrogenaseoxidoreductase domain, an aldehyde dehydrogenase family domain, a shortchain dehydrogenase domain, an oxidoreductase protein dehydrogenasedomain, a 3-beta hydroxysteroid dehydrogenase domain, a NAD-dependentepimerase/dehydratase domain, a short chain dehydrogenase/reductasedomain, a shikimate 5-dehydrogenase domain, a dehydrogenase domain,and/or a glucose-1-dehydrogenase domain.

[0017] In a preferred embodiment, a DHDR protein includes at least oneor more of the following domains: a transmembrane domain, a signalpeptide domain, an aldehyde dehydrogenase oxidoreductase domain, analdehyde dehydrogenase family domain, a short chain dehydrogenasedomain, an oxidoreductase protein dehydrogenase domain, a 3-betahydroxysteroid dehydrogenase domain, a NAD-dependentepimerase/dehydratase domain, a short chain dehydrogenase/reductasedomain, a shikimate 5-dehydrogenase domain, a dehydrogenase domain,and/or a glucose-1-dehydrogenase domain, and has an amino acid sequenceat least about 50%, 55%, 60%, 65%, 67%, 68%, 70%, 75%, 80%, 85%, 90%92%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,99.7%, 99.8%, 99.9%, 99.99% or more identical to the amino acid sequenceof SEQ ID NO:2, 5, 8, 11, or 15, or the amino acid sequence encoded bythe DNA insert of the plasmid deposited with ATCC as Accession Number______, PTA-1845, ______, ______, or ______.

[0018] In another preferred embodiment, a DHDR protein includes at leastone or more of the following domains: a transmembrane domain, a signalpeptide domain, an aldehyde dehydrogenase oxidoreductase domain, analdehyde dehydrogenase family domain, a short chain dehydrogenasedomain, an oxidoreductase protein dehydrogenase domain, a 3-betahydroxysteroid dehydrogenase domain, a NAD-dependentepimerase/dehydratase domain, a short chain dehydrogenase/reductasedomain, a shikimate 5-dehydrogenase domain, a dehydrogenase domain,and/or a glucose-1-dehydrogenase domain, and has a DHDR activity (asdescribed herein).

[0019] In yet another preferred embodiment, a DHDR protein includes atleast one or more of the following domains: a transmembrane domain, asignal peptide domain, an aldehyde dehydrogenase oxidoreductase domain,an aldehyde dehydrogenase family domain, a short chain dehydrogenasedomain, an oxidoreductase protein dehydrogenase domain, a 3-betahydroxysteroid dehydrogenase domain, a NAD-dependentepimerase/dehydratase domain, a short chain dehydrogenase/reductasedomain, a shikimate 5-dehydrogenase domain, a dehydrogenase domain,and/or a glucose-1-dehydrogenase domain, and is encoded by a nucleicacid molecule having a nucleotide sequence which hybridizes understringent hybridization conditions to a complement of a nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:1, 3, 4, 6, 7,9, 10, 12, 14, or 16.

[0020] In another embodiment, the invention features fragments of theprotein having the amino acid sequence of SEQ ID NO:2, 5, 8, 11, or 15,wherein the fragment comprises at least 16 amino acids (e g., contiguousamino acids) of the amino acid sequence of SEQ ID NO:2, 5, 8, 11, or 15,or an amino acid sequence encoded by the DNA insert of the plasmiddeposited with the ATCC as Accession Number ______, PTA-1845, ______,______, or ______. In another embodiment, a DHDR protein has the aminoacid sequence of SEQ ID NO:2, 5, 8, 11, or 15.

[0021] In another embodiment, the invention features a DHDR proteinwhich is encoded by a nucleic acid molecule consisting of a nucleotidesequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 89%,90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,99.7%, 99.8%, 99.9%, 99.99% or more identical to a nucleotide sequenceof SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16, or a complementthereof. This invention further features a DHDR protein which is encodedby a nucleic acid molecule consisting of a nucleotide sequence whichhybridizes under stringent hybridization conditions to a complement of anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1,3, 4, 6, 7, 9, 10, 12, 14, or 16, or a complement thereof.

[0022] The proteins of the present invention or portions thereof, e.g.,biologically active portions thereof, can be operatively linked to anon-DHDR polypeptide (e.g., heterologous amino acid sequences) to formfusion proteins. The invention further features antibodies, such asmonoclonal or polyclonal antibodies, that specifically bind proteins ofthe invention, preferably DHDR proteins. In addition, the DHDR proteinsor biologically active portions thereof can be incorporated intopharmaceutical compositions, which optionally include pharmaceuticallyacceptable carriers.

[0023] In another aspect, the present invention provides a method fordetecting the presence of a DHDR nucleic acid molecule, protein, orpolypeptide in a biological sample by contacting the biological samplewith an agent capable of detecting a DHDR nucleic acid molecule,protein, or polypeptide such that the presence of a DHDR nucleic acidmolecule, protein or polypeptide is detected in the biological sample.

[0024] In another aspect, the present invention provides a method fordetecting the presence of DHDR activity in a biological sample bycontacting the biological sample with an agent capable of detecting anindicator of DHDR activity such that the presence of DHDR activity isdetected in the biological sample.

[0025] In another aspect, the invention provides a method for modulatingDHDR activity comprising contacting a cell capable of expressing DHDRwith an agent that modulates DHDR activity such that DHDR activity inthe cell is modulated. In one embodiment, the agent inhibits DHDRactivity. In another-embodiment, the agent stimulates DHDR activity. Inone embodiment, the agent is an antibody that specifically binds to aDHDR protein. In another embodiment, the agent modulates expression ofDHDR by modulating transcription of a DHDR gene or translation of a DHDRmRNA. In yet another embodiment, the agent is a nucleic acid moleculehaving a nucleotide sequence that is antisense to the coding strand of aDHDR mRNA or a DHDR gene.

[0026] In one embodiment, the methods of the present invention are usedto treat a subject having a disorder characterized by aberrant orunwanted DHDR protein or nucleic acid expression or activity byadministering an agent which is a DHDR modulator to the subject. In oneembodiment, the DHDR modulator is a DHDR protein. In another embodimentthe DHDR modulator is a DHDR nucleic acid molecule. In yet anotherembodiment, the DHDR modulator is a peptide, peptidomimetic, or othersmall molecule. In a preferred embodiment, the disorder characterized byaberrant or unwanted DHDR protein or nucleic acid expression is adehydrogenase-associated disorder, e.g., a viral disorder, a CNSdisorder, a cardiovascular disorder, a muscular disorder, or a cellproliferation, growth, differentiation, or migration disorder.

[0027] The present invention also provides diagnostic assays foridentifying the presence or absence of a genetic alterationcharacterized by at least one of (i) aberrant modification or mutationof a gene encoding a DHDR protein; (ii) mis-regulation of the gene; and(iii) aberrant post-translational modification of a DHDR protein,wherein a wild-type form of the gene encodes a protein with a DHDRactivity.

[0028] In another aspect the invention provides methods for identifyinga compound that binds to or modulates the activity of a DHDR protein, byproviding an indicator composition comprising a DHDR protein having DHDRactivity, contacting the indicator composition with a test compound, anddetermining the effect of the test compound on DHDR activity in theindicator composition to identify a compound that modulates the activityof a DHDR protein.

[0029] Other features and advantages of the invention will be apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIGS. 1A-1D depict the cDNA sequence and predicted amino acidssequence of human DHDR-1 (clone FBH32142). The nucleotide sequencecorresponds to nucleic acids 1 to 2660 of SEQ ID NO:1. The amino acidsequence corresponds to amino acids 1 to 802 of SEQ ID NO:2. The codingregion without the 3′ untranslated region of the human DHDR-1 gene isshown in SEQ ID NO:3.

[0031]FIG. 2 depicts a structural, hydrophobicity, and antigenicityanalysis of the human DHDR-1 protein.

[0032]FIG. 3 depicts the results of a search which was performed againstthe MEMSAT database and which resulted in the identification of one“transmembrane domains” in the human DHDR-1 protein (SEQ ID NO:2).

[0033]FIG. 4 depicts the results of a search which was performed againstthe HMM database and which resulted in the identification of an“aldehyde dehydrogenase family domain” in the human DHDR-1 protein.

[0034] FIGS. 5A-5B depict the results of a search which was performedagainst the ProDom database and which resulted in the identification ofa “aldehyde dehydrogenase oxidoreductase domain” in the human DHDR-1protein (SEQ ID NO:2).

[0035] FIGS. 6A-6B depict the cDNA sequence and predicted amino acidsequence of human DHDR-2 (clone Fbh21481). The nucleotide sequencecorresponds to nucleic acids 1379 of SEQ ID NO:4. The amino acidsequence corresponds to amino acids 1 to 311 of SEQ ID NO:5. The codingregion without the 3′ untranslated region of the human DHDR-2 gene isshown in SEQ ID NO:6.

[0036]FIG. 7 depicts a structural, hydrophobicity, and antigenicityanalysis of the human DHDR-2 protein.

[0037]FIG. 8 depicts the results of a signal peptide prediction and asearch which was performed against the MEMSAT database and whichresulted in the identification of a signal peptide and one“transmembrane domain” in the human DHDR-2 protein (SEQ ID NO:5).

[0038]FIG. 9 depicts the results of a search which was performed againstthe HMM database and which resulted in the identification of a“short-chain dehydrogenase domain” in the human DHDR-2 protein.

[0039]FIG. 10 depicts the results of a search which was performedagainst the ProDom database and which resulted in the identification ofa “oxidoreductase protein dehydrogenase domain” in the human DHDR-2protein (SEQ ID NO:5).

[0040] FIGS. 11A-11B depict the cDNA sequence and predicted amino acidsequence of human DHDR-3 (clone Fbh25964). The nucleotide sequencecorresponds to nucleic acids 1 to 1725 of SEQ ID NO:7 The amino acidsequence corresponds to amino acids 1 to 369 of SEQ ID NO:8. The codingregion without the 3′ untranslated region of the human DHDR-3 gene isshown in SEQ ID NO:9.

[0041]FIG. 12 depicts a structural, hydrophobicity, and antigenicityanalysis of the human DHDR-3 protein.

[0042]FIG. 13 depicts the results of a search which was performedagainst the MEMSAT database and which resulted in the identification offour “transmembrane domains” in the human DHDR-3 protein (SEQ ID NO:8).

[0043] FIGS. 14A-14B depicts the results of a search which was performedagainst the HMM database and which resulted in the identification of a“3-beta hydroxysteroid dehydrogenase domain”, a “short chaindehydrogenase domain”, and a “NAD-dependent epimerase/dehydratasedomain” in the human DHDR-3 protein.

[0044]FIG. 15 depicts the results of a search which was performedagainst the ProDom database and which resulted in the identification ofa “3-beta hydroxysteroid dehydrogenase domain” in the human DHDR-3protein (SEQ ID NO:8).

[0045]FIG. 16 depicts the cDNA sequence and predicted amino acidsequence of human DHDR-4 (clone Fbh21686). The nucleotide sequencecorresponds to nucleic acids 1 to 1209 of SEQ ID NO:10. The amino acidsequence corresponds to amino acids 1 to 322 of SEQ ID NO:11. The codingregion without the 3′ untranslated region of the human DHDR-4 gene isshown in SEQ ID NO:12.

[0046]FIG. 17 depicts an alignment of the human DHDR-4 amino acidsequence (“21686”; SEQ ID NO:11) with the amino acid sequence of Rattusnorvegicus putative short-chain dehydrogenase/reductase(“5052204_SDR_rat”; GenBank Accession Number AF099742; SEQ ID NO:13)using the CLUSTAL W (1.74) multiple sequence alignment program.Identical amino acids are indicated by stars.

[0047]FIG. 18 depicts a structural, hydrophobicity, and antigenicityanalysis of the human DHDR-4 protein.

[0048]FIG. 19 depicts the results of a signal peptide prediction and asearch which was performed against the MEMSAT database and whichresulted in the identification of a “signal peptide” and four“transmembrane domains” in the human DHDR-4 protein (SEQ ID NO:11).

[0049]FIG. 20 depicts the results of a search which was performedagainst the HMM database and which resulted in the identification of a“short chain dehydrogenase domain” and a “short chaindehydrogenase/reductase domain” in the human DHDR-4 protein.

[0050] FIGS. 21A-21B depict the results of a search which was performedagainst the ProDom database and which resulted in the identification ofa “oxidoreductase protein dehydrogenase domain”, a “shikimate5-dehydrogenase domain”, a “dehydrogenase domain” and a“glucose-1-dehydrogenase domain” in the human DHDR-4 protein (SEQ IDNO:11).

[0051]FIG. 22 depicts the expression levels of human DHDR-1 mRNA invarious human cell types and tissues, as determined by Taqman analysis.Samples: (1) normal artery; (2) normal vein; (3) aortic smooth musclecells—early; (4) coronary smooth muscle cells; (5) human microvascularendothelial cells (HMVECs)—static; (6) human microvascular endothelialcells (HMVECs)—shear; (7) normal heart; (8) heart—congestive heartfailure (CHF); (9) kidney; (10) skeletal muscle; (11) normal adiposetissue; (12) pancreas; (13) primary osteoblasts; (14) differentiatedosteoclasts; (15) normal skin; (16) normal spinal cord; (17) normalbrain cortex; (18) brain—hypothalamus; (19) nerve; (20) dorsal rootganglion (DRG); (21) resting peripheral blood mononuclear cells (PBMCs);(22) glioblastoma; (23) normal breast; (24) breast tumor; (25) normalovary; (26) ovary tumor; (27) normal prostate; (28) prostate tumor; (29)epithelial cells (prostate); (30) normal colon; (31) colon tumor; (32)normal lung; (33) lung tumor; (34) lung—chronic obstructive pulmonarydisease (COPD); (35) colon—inflammatory bowel disease (IBD); (36) normalliver; (37) liver—fibrosis; (38) dermal cells—fibroblasts; (39) normaltonsil; (40) lymph node; (41) small intestine; (42) skin—decubitus; (43)synovium; (44) bone marrow mononuclear cells (BM-MNC); (45) activatedperipheral blood mononuclear cells (PBMCs).

[0052]FIG. 23 depicts the expression levels of human DHDR-1 mRNA invarious types of human tumors, as determined by Taqman analysis.Samples: (1-3) normal breast; (4) breast tumor—infiltrating ductalcarcinoma (IDC); (5) breast tumor—infiltrating ductal carcinoma(MD-IDC); (6-8) breast tumor—infiltrating ductal carcinoma (IDC); (9)breast tumor; (10-11) normal ovary; (12-16) ovary tumor; (17-19) normallung; (20) lung tumor—SmC; (21-23) lung tumor—poorly differentiatednon-small cell carcinoma of the lung (PDNSCCL); (24) lung tumor—smallcell carcinoma (SCC); (25) lung tumor—AC; (26) lung tumor—ACA; (27-29)normal colon; (30-31) colon tumor—MD; (32) colon tumor; (33) colontumor—MD-PD; (34-35) colon tumor—liver metastasis; (36) normal liver(female); (37) hemangioma; (38) human microvascular endothelial cells(HMVECs)—arrested; (39) human microvascular endothelial cells(HMVECs)—proliferating.

[0053]FIG. 24 depicts the expression levels of human DHDR-1 mRNA invarious human colon tumor samples, as determined by Taqman analysis.Samples: (1-6) normal colon; (7-8) adenomas; (9-15) colonic ACA-B;(16-21); colonic ACA-C; (22-27) normal liver; (28-33) colon tumor—livermetastasis; (34) colon tumor—abdominal metastasis.

[0054]FIG. 25 depicts the expression levels of human DHDR-1 mRNA in NOCsynchronized HCT116 cells at various time points after entry into thecell cycle, as determined by Taqman analysis. The time point t=0signifies the G2/M border. Samples: (1) t=0; (2) t=3; (3) t=6; (4) t=9;(5) t=15; (6) t=18; (7) t=21; (8) t=24.

[0055]FIG. 26 depicts the expression levels of human DHDR-2 mRNA invarious human clinical tumor samples, as determined by Taqman analysis.Samples: (1-4) normal breast; (5-11) breast tumor; (12-14) normal lung;(15-22) lung tumor; (23-25) normal colon; (26-33) colon tumor; (34-37)colon tumor—liver metastasis; (38-39) normal liver; (40) normal brain;(41-43) brain tumor—glioblastoma.

[0056]FIG. 27 depicts the expression levels of human DHDR-4 mRNA invarious human cell types and tumors, as determined by Taqman analysis.Samples: (1) normal aorta; (2) normal fetal heart; (3) normal heart; (4)heart—congestive heart failure (CHF); (5) normal vein; (6) aortic smoothmuscle cells; (7) normal spinal cord; (8) normal brain cortex; (9)brain—hypothalamus; (10) glial cells—astrocytes; (11)brain—glioblastoma; (12) normal breast; (13) breast tumor—infiltratingductal carcinoma (IDC); (14) normal ovary; (15) ovary tumor; (16)pancreas; (17) normal prostate; (18) prostate tumor; (19) normal colon;(20) colon tumor; (21) colon—inflammatory bowel disease (IBD); (22)normal kidney; (23) normal liver; (24) liver—fibrosis; (25) normal fetalliver; (26) normal lung; (27) lung tumor; (28) lung—chronic obstructivepulmonary disease (COPD); (29) normal spleen; (30) normal tonsil; (31)normal lymph node; (32) normal thymus; (33) epithelial cells—prostate;(34) endothelial cells—aortic; (35) skeletal muscle; (36) dermalfibroblasts; (37) normal skin; (38) normal adipose tissue; (39) primaryosteoblasts; (40) undifferentiated osteoblasts; (41) differentiatedosteoblasts; (42) osteoclasts; (43) aortic smooth muscle cells(SMCs)—early; (44) aortic smooth muscle cells (SMCs)—late; (45) humanumbilical vein endothelial cells (HUVECs)—shear; (46) human umbilicalvein endothelial cells (HUVECs)—static.

[0057]FIG. 28 depicts the expression levels of human DHDR-4 mRNA invarious cell types and tissues, as determined by Taqman analysis.Samples: (1-2) normal liver; (3-4) HBV+ liver; (5) HCV+ liver; (6)HepG2-B cells; (7) HepG2.2.15-B cells; (8) HepG2 cells (no treatment);(9) HepG2 cells—treated with Bayer compound (IC50); (10) HepG2cells—treated with Bayer compound (IC100); (11) HepG2.2.15 cells (notreatment); (12) HepG2.2.15 cells—treated with Bayer compound (IC50);(13) HepG2.2.15 cells—treated with Bayer compound (IC100); (14) HepG2control; (15) HepG2 cells transfected with the HBV-X gene; (16) HuH7cells; (17-19) ganglia; (20) NT2/KOS—0 hr.; (21) NT2/KOS—2.5 hr.; (22)NT2/KOS—5 hr.; (23) NT2/KOS—7 hr.; (24) MRC/VZV—mock; (25) MRC/VZV —18hr.; (26) MRC/VZV—72 hr.

[0058]FIG. 29 depicts the expression levels of human DHDR-4 mRNA invarious human tumor samples, as determined by Taqman analysis. Samples:(1-4) normal colon; (5-11) colon tumor; (12-15) colon tumor—livermetastasis; (16-17) normal liver; (18-21) normal brain; (22-27) braintumor—glioblastoma; (28-29) human microvascular endothelial cells(HMVECs); (30) placenta; (31-32) fetal adrenal gland; (33-34) fetalliver.

[0059]FIG. 30 depicts the expression levels of human DHDR-4 mRNA invarious human tumors and synchronized A549 cells at various time pointsafter entry into the cell cycle, as determined by Taqman analysis. Thetime point t=0 signifies the G2/M border. Samples: (1-4) normal breast;(5-10) breast tumor; (11-14) normal ovary; (15-22) ovary tumor; (23-26)normal lung; (27-34) lung tumor; (35-42) synchronized A549 cells: (35) t=0; (36) t=3 (RT); (37) t=3 (−RT); (38) t=6; (39) t=9; (40) t=12 (RT);(41) t=18; (42) t=24.

[0060] FIGS. 31A-31B depict the cDNA sequence and predicted amino acidsequence of mouse DHDR-2 (clone m21481). The nucleotide sequence,corresponding to nucleic acids 1 to 1108 of SEQ ID NO:14, is shown inFIG. 31A. The coding region, corresponding to SEQ ID NO:16, isunderlined. The amino acid sequence, corresponding to amino acids 1 to311 of SEQ ID NO:15, is shown in FIG. 31B.

[0061] FIGS. 32A-32B depict an alignment of the mouse DHDR-2 nucleotidesequence (“M21484”, SEQ ID NO:14) with the human DHDR-2 nucleotidesequence (“h21484”; SEQ ID NO:4) using the GAP program in the GCGsoftware package (nwsgapdna.cmp matrix) and a gap weight of 12 and alength weight of 4. As shown in the alignment, the mouse and humanDHDR-2 nucleotide sequences are about 88.1% identical.

[0062]FIG. 33 depicts an alignment of the mouse DHDR-2 amino acidsequence (“m21484”; SEQ ID NO:15) with the human DHDR-2 amino acidsequence (“h21484”; SEQ ID NO:5) using the GAP program in the GCGsoftware package (Blosum 62 matrix) and a gap weight of 12 and a lengthweight of 4. As shown in the alignment, the mouse and human DHDR-2 aminoacid sequences are about 91.3% identical.

DETAILED DESCRIPTION OF THE INVENTION

[0063] The present invention is based, at least in part, on thediscovery of novel molecules, referred to herein as “dehydrogenase” or“DHDR” nucleic acid and protein molecules, which are novel members of afamily of enzymes possessing dehydrogenase activity. These novelmolecules are capable of oxidizing or reducing biological molecules bycatalyzing the transfer of a hydride moiety and, thus, play a role in orfunction in a variety of cellular processes, e.g., proliferation,growth, differentiation, migration, immune responses, hormonalresponses, inter- or intra-cellular communication, and viral infection.

[0064] As used herein, the term “dehydrogenase” includes a moleculewhich is involved in the oxidation or reduction of a biochemicalmolecule (e.g., an amino acid, a vitamin, a steroid such as aglucocorticoid, or a nucleic acid), by catalyzing the transfer of ahydride ion to or from the biochemical molecule. Dehydrogenase moleculesare involved in the metabolism and catabolism of biochemical moleculesnecessary for energy production or storage, for intra- or inter-cellularsignaling, for metabolism or catabolism of metabolically importantbiomolecules, and for detoxification of potentially harmful compounds.Examples of dehydrogenases include alcohol dehydrogenases, aldehydedehydrogenases, steroid dehydrogenases, and lipid dehydrogenases. Thus,the DHDR molecules of the present invention provide novel diagnostictargets and therapeutic agents to control dehydrogenase-associateddisorders.

[0065] As used herein, a “dehydrogenase-associated disorder” includes adisorder, disease or condition which is caused or characterized by amisregulation (e.g., downregulation or upregulation) of dehydrogenaseactivity. Dehydrogenase-associated disorders can detrimentally affectcellular functions such as cellular proliferation, growth,differentiation, or migration, inter- or intra-cellular communication;tissue function, such as cardiac function or musculoskeletal function;systemic responses in an organism, such as nervous system responses,hormonal responses (e.g., insulin response), susceptibility topathogenic infections (e.g., viral infections), or immune responses; andprotection of cells from toxic compounds (e.g., carcinogens, toxins, ormutagens). Examples of dehydrogenase-associated disorders include CNSdisorders such as cognitive and neurodegenerative disorders, examples ofwhich include, but are not limited to, Alzheimer's disease, dementiasrelated to Alzheimer's disease (such as Pick's disease), Parkinson's andother Lewy diffuse body diseases, senile dementia, Huntington's disease,Gilles de la Tourette's syndrome, multiple sclerosis, amyotrophiclateral sclerosis, progressive supranuclear palsy, epilepsy, andJakob-Creutzfieldt disease; autonomic function disorders such ashypertension and sleep disorders, and neuropsychiatric disorders, suchas depression, schizophrenia, schizoaffective disorder, korsakoff'spsychosis, mania, anxiety disorders, or phobic disorders; learning ormemory disorders, e.g., amnesia or age-related memory loss, attentiondeficit disorder, dysthymic disorder, major depressive disorder, mania,obsessive-compulsive disorder, psychoactive substance use disorders,anxiety, phobias, panic disorder, as well as bipolar affective disorder,e.g., severe bipolar affective (mood) disorder (BP-1), and bipolaraffective neurological disorders, e.g., migraine and obesity. FurtherCNS-related disorders include, for example, those listed in the AmericanPsychiatric Association's Diagnostic and Statistical manual of MentalDisorders (DSM), the most current version of which is incorporatedherein by reference in its entirety.

[0066] Further examples of dehydrogenase-associated disorders includecardiac-related disorders. Cardiovascular system disorders in which theDHDR molecules of the invention may be directly or indirectly involvedinclude arteriosclerosis, ischemia reperfusion injury, restenosis,arterial inflammation, vascular wall remodeling, ventricular remodeling,rapid ventricular pacing, coronary microembolism, tachycardia,bradycardia, pressure overload, aortic bending, coronary arteryligation, vascular heart disease, atrial fibrilation, Jervell syndrome,Lange-Nielsen syndrome, long-QT syndrome, congestive heart failure,sinus node dysfunction, angina, heart failure, hypertension, atrialfibrillation, atrial flutter, dilated cardiomyopathy, idiopathiccardiomyopathy, myocardial infarction, coronary artery disease, coronaryartery spasm, and arrhythmia. DHDR-mediated or related disorders alsoinclude disorders of the musculoskeletal system such as paralysis andmuscle weakness, e.g., ataxia, myotonia, and myokymia.

[0067] Dehydrogenase-associated disorders also include cellularproliferation, growth, differentiation, or migration disorders. Cellularproliferation, growth, differentiation, or migration disorders includethose disorders that affect cell proliferation, growth, differentiation,or migration processes. As used herein, a “cellular proliferation,growth, differentiation, or migration process” is a process by which acell increases in number, size or content, by which a cell develops aspecialized set of characteristics which differ from that of othercells, or by which a cell moves closer to or further from a particularlocation or stimulus. The DHDR molecules of the present invention areinvolved in signal transduction mechanisms, which are known to beinvolved in cellular growth, differentiation, and migration processes.Thus, the DHDR molecules may modulate cellular growth, differentiation,or migration, and may play a role in disorders characterized byaberrantly regulated growth, differentiation, or migration. Suchdisorders include cancer, e.g., carcinomas, sarcomas, leukemias, andlymphomas; tumor angiogenesis and metastasis; skeletal dysplasia;hepatic disorders; and hematopoietic and/or myeloproliferativedisorders.

[0068] DHDR-associated or related disorders also include hormonaldisorders, such as conditions or diseases in which the production and/orregulation of hormones in an organism is aberrant. Examples of suchdisorders and diseases include type I and type II diabetes mellitus,pituitary disorders (e.g., growth disorders), thyroid disorders (e.g.,hypothyroidism or hyperthyroidism), and reproductive or fertilitydisorders (e.g., disorders which affect the organs of the reproductivesystem, e.g., the prostate gland, the uterus, or the vagina; disorderswhich involve an imbalance in the levels of a reproductive hormone in asubject; disorders affecting the ability of a subject to reproduce; anddisorders affecting secondary sex characteristic development, e.g.,adrenal hyperplasia).

[0069] DHDR-associated or related disorders also include immunedisorders, such as autoimmune disorders or immune deficiency disorders,e.g., allergies, transplant rejection, responses to pathogenic infection(e.g., bacterial, viral, or parasitic infection), lupus, multiplesclerosis, congenital X-linked infantile hypogammaglobulinemia,transient hypogammaglobulinemia, common variable immunodeficiency,selective IgA deficiency, chronic mucocutaneous candidiasis, or severecombined immunodeficiency.

[0070] DHDR-associated or related disorders also include viraldisorders, i.e., disorders affected or caused by infection by viruses(e.g., hepatitis A, hepatitis B, hepatitis delta, and otherhepadnaviruses; Coxsackie B viruses; Epstein-Barr virus; adenovirus;rhinoviruses; human immunodeficiency virus (HIV); vaccinia virus; humanT cell leukemia virus; RD114 virus; herpes simplex, herpes zoster, andother herpesviruses; Marek's disease virus; Yamaguchi sarcoma virus;human papillomaviruses; poliovirus; poxviruses; influenza virus;cytomegalovirus; encephalitis viruses; measles viruses; and ebola andother hemorrhagic viruses). Such disorders include, but are not limitedto, hepatocellularcarcinoma, cirrhosis of the liver, cervical carcinoma,Burkitt's lymphoma, lymphoproliferative disease, Kaposi's sarcoma, Tcell leukemia, B cell lymphoma, plasmablastic lymphoma, Rasmussen'ssyndrome, Marek's disease, warts (including common, genital, and plantarwarts), genital herpes, common colds, acquired immune deficiencysyndrome (AIDS), polymyositis, immunorestitution disease, chicken pox,shingles, ebola and other hemorrhagic fever diseases, cold sores,transient or acute hepatitis, chronic hepatitis, influenza, Reyesyndrome, measles, Paget's disease, viral encephalitis, viral pneumonia,and viral meningitis.

[0071] Viral disorders also include disorders or conditions influencedby virus or viral activity. As used interchangeably herein, the terms“viral activity” and “virus activity” includes any activity known in theart to be characteristic of a virus and which can be detected by methodsknown in the art. For example, viral activity includes, but it notlimited to, the ability of a virus to infect a cell or an organism(e.g., a human), the ability of a virus to replicate or reproduce in acell or an organism (e.g., a human), the ability of a virus to induce animmune response in vitro or in vivo, the ability to induce expression ofviral genes, and/or the ability to induce expression of host orexogenous genes not normally expressed by an uninfected cell.

[0072] DHDR-associated or related disorders also include disordersaffecting tissues in which DHDR protein is expressed, e.g., liver,hepatocytes, hepatitis B-infected hepatocytes, HepG2 cells, hepatitisB-infected HepG2.2.15 cells, kidney, brain, primary osteoblasts,pituitary, CaCO cells, keratinocytes, aortic endothelial cells, fetalkidney, fetal lung, mammary epithelium, fetal spleen, fetal liver,umbilical smooth muscle, RAII Burkitt Lymphoma cells, lung, prostate,K53 red blood cells, fetal dorsal spinal cord, insulinoma cells, normalbreast and ovarian epithelia, retina, HMC-1 mast cells, ovarian ascites,d8 dendritic cells, megakaryocytes, human mobilized bone morrow, mammarycarcinoma, melanoma cells, lymph, vein, U937/A70p B cells, A549concells, WT LN Cap testosterone cells, and esophagus.

[0073] As used herein, a “dehydrogenase-mediated activity” includes anactivity which involves the oxidation or reduction of one or morebiochemical molecules, e.g., biochemical molecules (e.g.,glucocorticoids) in a neuronal cell, a liver cell, or a tumor cellassociated with the regulation of one or more cellular processes.Dehydrogenase-mediated activities include the oxidation or reduction ofbiochemical molecules necessary for energy production or storage, forintra- or inter-cellular signaling, for metabolism or catabolism ofmetabolically important biomolecules, for viral infection, and fordetoxification of potentially harmful compounds.

[0074] The term “family” when referring to the protein and nucleic acidmolecules of the invention is intended to mean two or more proteins ornucleic acid molecules having a common structural domain or motif andhaving sufficient amino acid or nucleotide sequence homology as definedherein. Such family members can be naturally or non-naturally occurringand can be from either the same or different species. For example, afamily can contain a first protein of human origin, as well as other,distinct proteins of human origin or alternatively, can containhomologues of non-human origin, e.g., monkey proteins. Members of afamily may also have common functional characteristics.

[0075] For example, the family of DHDR proteins comprises at least one“transmembrane domain”. As used herein, the term “transmembrane domain”includes an amino acid sequence of about 15 amino acid residues inlength which spans the plasma membrane. More preferably, a transmembranedomain includes about at least 20, 25, 30, 35, 40, or 45 amino acidresidues and spans the plasma membrane. Transmembrane domains are richin hydrophobic residues, and typically have an alpha-helical structure.In a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or moreof the amino acids of a transmembrane domain are hydrophobic, e.g.,leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domainsare described in, for example, Zagotta W. N. et al., (1996) Annu. Rev.Neurosci. 19:235-263, the contents of which are incorporated herein byreference. Amino acid residues 159-175 of the native DHDR-1 protein arepredicted to comprise a transmembrane domain (see FIG. 3). Amino acidresidues 7-23 of the native DHDR-2 protein and residues 265-283 of themature DHDR-2 protein are predicted to comprise a transmembrane domain(see FIG. 8). Amino acid residues 10-26, 73-90, 289-305, and 312-333 ofthe native DHDR-3 protein are predicted to comprise transmembranedomains (see FIG. 13). Amino acid residues 29-50, 170-188, 108-224, and258-275 of the native DHDR-4 protein and residues 10-31, 151-169,189-205, and 239-256 of the mature DHDR-4 protein are predicted tocomprise transmembrane domains (see FIG. 19). Accordingly, DHDR proteinshaving at least 50-60% homology, preferably about 60-70%, morepreferably about 70-80%, or about 80-90% homology with a transmembranedomain of human DHDR are within the scope of the invention.

[0076] In another embodiment of the invention, a DHDR protein of thepresent invention is identified based on the presence of a signalpeptide. The prediction of such a signal peptide can be made, forexample, utilizing the computer algorithm SignalP (Henrik et al. (1997)Prot. Eng. 10:1-6). As used herein, a “signal sequence” or “signalpeptide” includes a peptide containing about 15 or more amino acidswhich occurs at the N-terminus of secretory and membrane bound proteinsand which contains a large number of hydrophobic amino acid residues.For example, a signal sequence contains at least about 10-30 amino acidresidues, preferably about 15-25 amino acid residues, more preferablyabout 18-20 amino acid residues, and more preferably about 19 amino acidresidues, and has at least about 35-65%, preferably about 38-50%, andmore preferably about 40-45% hydrophobic amino acid residues (e.g.,Valine, Leucine, Isoleucine or Phenylalanine). Such a “signal sequence”,also referred to in the art as a “signal peptide”, serves to direct aprotein containing such a sequence to a lipid bilayer, and is cleaved insecreted and membrane bound proteins. A signal sequence was identifiedin the amino acid sequence of human DHDR-2 at about amino acids 1-18 ofSEQ ID NO:5. A signal sequence was also identified in the amino acidsequence of human DHDR-4 at about amino acids 1-19 of SEQ ID NO:11.

[0077] In another embodiment, a DHDR molecule of the present inventionis identified based on the presence of an “aldehyde dehydrogenase familydomain” in the protein or corresponding nucleic acid molecule. As usedherein, the term “aldehyde dehydrogenase family domain” includes aprotein domain having an amino acid sequence of about 350-550 amino acidresidues and a bit score of at least 149.8. Preferably, an aldehydedehydrogenase family domain includes at least about 400-500, or morepreferably about 448 amino acid residues, and a bit score of about 50,60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,210, 220, 230, 240, or 250 or more. To identify the presence of analdehyde dehydrogenase family domain in a DHDR protein, and make thedetermination that a protein of interest has a particular profile, theamino acid sequence of the protein is searched against a database ofknown protein domains (e.g., the HMM database). The aldehydedehydrogenase family domain (HMM) has been assigned the PFAM AccessionPF00171 (see the Pfam website, available online through WashingtonUniversity in Saint Louis). A search was performed against the HMMdatabase-resulting in the identification of an aldehyde dehydrogenasefamily domain in the amino-acid sequence of human DHDR-1 (SEQ ID NO:2)at about residues 47-494 of SEQ ID NO:2. The results of the search areset forth in FIG. 4.

[0078] In another embodiment, a DHDR molecule of the present inventionis identified based on the presence of an “aldehyde dehydrogenaseoxidoreductase domain” in the protein or corresponding nucleic acidmolecule. As used herein, the term “aldehyde dehydrogenaseoxidoreductase domain” includes a protein domain having an amino acidsequence of about 550-750 amino acid residues and having a bit score forthe alignment of the sequence to the aldehyde dehydrogenaseoxidoreductase domain of at least 280. Preferably, an aldehydedehydrogenase oxidoreductase domain includes at least about 600-700, ormore preferably about 670 amino acid residues, and has a bit score forthe alignment of the sequence to the aldehyde dehydrogenaseoxidoreductase domain of at least 125, 150, 175, 200, 225, 250, 275,300, 325, 350, 375, 400 or higher. The aldehyde dehydrogenaseoxidoreductase domain has been assigned ProDom entry 135. To identifythe presence of an aldehyde dehydrogenase oxidoreductase domain in aDHDR protein, and to make the determination that a protein of interesthas a particular profile, the amino acid sequence of the protein issearched against a database of known protein domains (e.g., the ProDomdatabase) using the default parameters (available online through theProDom website). A search was performed against the ProDom databaseresulting in the identification of an aldehyde dehydrogenaseoxidoreductase domain in the amino acid sequence of human DHDR-1 (SEQ IDNO:2) at about residues 101-770 of SEQ ID NO:2. The results of thesearch are set forth in FIGS. 5A-5B.

[0079] In another embodiment, a DHDR molecule of the present inventionis identified based on the presence of a “short chain dehydrogenasedomain” in the protein or corresponding nucleic acid molecule. As usedherein, the term “short chain dehydrogenase domain” includes a proteindomain having an amino acid sequence of about 100-300 amino acidresidues, and a bit score of at least 120.0-162.5. Preferably, a shortchain dehydrogenase domain includes at least about 150-250, or morepreferably about 187-195 amino acid residues, and has a bit score of atleast 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200,210, 220, or more. To identify the presence of a shortchain dehydrogenase domain in a DHDR protein, and make the determinationthat a protein of interest has a particular profile, the amino acidsequence of the protein is searched against a database of known proteindomains (e.g., the HMM database). The short chain dehydrogenase domain(HMM) has been assigned the PFAM Accession PF00106 (see the Pfamwebsite, available online through Washington University in Saint Louis).A search was performed against the HMM database resulting in theidentification of a short chain dehydrogenase domain in the amino acidsequence of human DHDR-2 (SEQ ID NO:5) at about residues 38-227 of SEQID NO:5. The results of the search are set forth in FIG. 9. A search wasalso performed against the HMM database resulting in the identificationof a short chain dehydrogenase domain in the amino acid sequence ofhuman DHDR-3 (SEQ ID NO:8) at about residues 10-197 of SEQ ID NO:8. Theresults of this search are set forth in FIGS. 14A-14B. Another searchperformed against the HMM database resulted in the identification of ashort chain dehydrogenase domain in the amino acid sequence of humanDHDR-4 (SEQ ID NO:11) at about residues 38-226 of SEQ ID NO:11. Theresults of this search are set forth in FIG. 20.

[0080] In another embodiment, a DHDR molecule of the present inventionis identified based on the presence of an “oxidoreductase proteindehydrogenase domain” in the protein or corresponding nucleic acidmolecule. As used herein, the term “oxidoreductase protein dehydrogenasedomain” includes a protein domain having an amino acid sequence of about50-300 amino acid residues and having a bit score for the alignment ofthe sequence to the oxidoreductase protein dehydrogenase domain of atleast 113. Preferably, an oxidoreductase protein dehydrogenase domainincludes at least about 100-250, or more preferably about 120-200 aminoacid residues, and has a bit score for the alignment of the sequence tothe oxidoreductase protein dehydrogenase domain of at least 40, 50, 60,70, 80, 35 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, or higher.The oxidoreductase protein dehydrogenase domain has been assigned ProDomentry 11. To identify the presence of an oxidoreductase proteindehydrogenase domain in a DHDR protein, and to make the determinationthat a protein of interest has a particular profile, the amino acidsequence of the protein is searched against a database of known proteindomains (e.g., the ProDom database) using the default parameters(available online through the ProDom website). A search was performedagainst the ProDom database resulting in the identification of anoxidoreductase protein dehydrogenase domain in the amino acid sequenceof human DHDR-2 (SEQ ID NO:5) at about residues 99-219 of SEQ ID NO:5.The results of the search are set forth in FIG. 10. Another search wasperformed against the ProDom database, resulting in the identificationof an oxidoreductase protein dehydrogenase domain in the amino acidsequence of human DHDR-4 (SEQ ID NO:11) at about residues 37-231 of SEQID NO:11. The results of this search are set forth in FIGS. 21 A-21B.

[0081] In another embodiment, a DHDR molecule of the present inventionis identified based on the presence of an “NAD-dependentepimerase/dehydratase domain” in the protein or corresponding nucleicacid molecule. As used herein, the term “NAD-dependentepimerase/dehydratase domain” includes a protein domain having an aminoacid sequence of about 250-450 amino acid residues. Preferably, anNAD-dependent epimerase/dehydratase domain includes at least about300-400, or more preferably about 354 amino acid residues. To identifythe presence of an NAD-dependent epimerase/dehydratase domain in a DHDRprotein, and to make the determination that a protein of interest has aparticular profile, the amino acid sequence of the protein is searchedagainst a database of known protein domains (e.g., the HMM database).The NAD-dependent epimerase/dehydratase domain (HMM) has been assignedthe PFAM Accession PF01370 (see the Pfam website, available onlinethrough Washington University in Saint Louis). A search was performedagainst the HMM database resulting in the identification of anNAD-dependent epimerase/dehydratase domain in the amino acid sequence ofhuman DHDR-3 (SEQ ID NO:8) at about residues 12-365 of SEQ ID NO:8. Theresults of the search are set forth in FIGS. 14A-14B.

[0082] In another embodiment, a DHDR molecule of the present inventionis identified based on the presence of a “3-beta hydroxysteroiddehydrogenase domain” in the protein or corresponding nucleic acidmolecule. As used herein, the term “3-beta hydroxysteroid dehydrogenasedomain” includes a protein domain having an amino acid sequence of about250-450 amino acid residues and having a bit score for the alignment ofthe sequence to the 3-beta hydroxysteroid dehydrogenase domain of atleast 395-676.9. Preferably, a 3-beta hydroxysteroid dehydrogenasedomain includes at least about 300-400, or more preferably about 352-365amino acid residues, and has a bit score for the alignment of thesequence to the 3-beta hydroxysteroid dehydrogenase domain of at least300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625,650, 675, 700, 725, 750, 775, 800, 825 or higher. The 3-betahydroxysteroid dehydrogenase domain has been assigned ProDom entry 1280.To identify the presence of a 3-beta hydroxysteroid dehydrogenase domainin a DHDR protein, and to make the determination that a protein ofinterest has a particular profile, the amino acid sequence of theprotein is searched against a database of known protein domains (e.g.,the ProDom database) using the default parameters (available onlinethrough the ProDom website). A search was performed against the ProDomdatabase resulting in the identification of a 3-beta hydroxysteroiddehydrogenase domain in the amino acid sequence of human DHDR-3 (SEQ IDNO:8) at about residues 11-362 of SEQ ID NO:8. The results of the searchare set forth in FIG. 15. A search was also performed against the HMMdatabase resulting in the identification of a 3-beta hydroxysteroiddehydrogenase domain (PFAM accession PF01073, see the Pfam website,available online through Washington University in Saint Louis) in theamino acid sequence of human DHDR-3 (SEQ ID NO:8) at about residues1-365 of SEQ ID NO:8. The results of the search are set forth in FIGS.14A-14B.

[0083] In another embodiment, a DHDR molecule of the present inventionis identified based on the presence of a “short-chaindehydrogenase/reductase domain” in the protein or corresponding nucleicacid molecule. As used herein, the term “short-chaindehydrogenase/reductase domain” includes a protein domain having anamino acid sequence of about 10-100 amino acid residues, and a bit scoreof at least 47.2. Preferably, a short-chain dehydrogenase/reductasedomain includes at least about 20-75, or more preferably about 31 aminoacid residues, and has a bit score of at least 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more. To identifythe presence of a short-chain dehydrogenase/reductase domain in a DHDRprotein, and to make the determination that a protein of interest has aparticular profile, the amino acid sequence of the protein is searchedagainst a database of known protein domains (e g., the HMM database).The short-chain dehydrogenase/reductase domain (HMM) has been assignedthe PFAM Accession PF00678 (see the Pfam website, available onlinethrough Washington University in Saint Louis). A search was performedagainst the HMM database resulting in the identification of ashort-chain dehydrogenase/reductase domain in the amino acid sequence ofhuman DHDR-4 (SEQ ID NO:11) at about residues 250-280 of SEQ ID NO:11.The results of the search are set forth in FIG. 20.

[0084] In another embodiment, a DHDR molecule of the present inventionis identified based on the presence of a “shikimate 5-dehydrogenasedomain” in the protein or corresponding nucleic acid molecule. As usedherein, the term “shikimate 5-dehydrogenase domain” includes a proteindomain having an amino acid sequence of about 10-100 amino acid residuesand having a bit score for the alignment of the sequence to theshikimate 5-dehydrogenase domain of at least 86. Preferably, a shikimate5-dehydrogenase domain includes at least about 25-75, or more preferablyabout 48 amino acid residues, and has a bit score for the alignment ofthe sequence to the shikimate 5-dehydrogenase domain of at least 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or higher. The shikimate5-dehydrogenase domain has been assigned ProDom entry 95301. To identifythe presence of a shikimate 5-dehydrogenase domain in a DHDR protein,and to make the determination that a protein of interest has aparticular profile, the amino acid sequence of the protein is searchedagainst a database of known protein domains (e.g., the ProDom database)using the default parameters (available online through the ProDomwebsite). A search was performed against the ProDom database resultingin the identification of a shikimate 5-dehydrogenase domain in the aminoacid sequence of human DHDR-4 (SEQ ID NO:11) at about residues 35-82 ofSEQ ID NO:11. The results of the search are set forth in FIGS. 21A-21B.

[0085] In another embodiment, a DHDR molecule of the present inventionis identified based on the presence of a “dehydrogenase domain” in theprotein or corresponding nucleic acid molecule. As used herein, the term“dehydrogenase domain” includes a protein domain having an amino acidsequence of about 10-100 amino acid residues and having a bit score forthe alignment of the sequence to the dehydrogenase domain of at least84. Preferably, a dehydrogenase domain includes at least about 25-75, ormore preferably about 50 amino acid residues, and has a bit score forthe alignment of the sequence to the dehydrogenase domain of at least20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or higher. Thedehydrogenase domain has been assigned ProDom entry 73753. To identifythe presence of a dehydrogenase domain in a DHDR protein, and to makethe determination that a protein of interest has a particular profile,the amino acid sequence of the protein is searched against a database ofknown protein domains (e.g., the ProDom database) using the defaultparameters (available online through the ProDom website). A search wasperformed against the ProDom database resulting in the identification ofa dehydrogenase domain in the amino acid sequence of human DHDR-4 (SEQID NO:11) at about residues 237-286 of SEQ ID NO:11. The results of thesearch are set forth in FIGS. 21A-21B.

[0086] In another embodiment, a DHDR molecule of the present inventionis identified based on the presence of a “glucose-1-dehydrogenasedomain” in the protein or corresponding nucleic acid molecule. As usedherein, the term “glucose-1-dehydrogenase domain” includes a proteindomain having an amino acid sequence of about 10-100 amino acid residuesand having a bit score for the alignment of the sequence to theglucose-1-dehydrogenase domain of at least 92. Preferably, adehydrogenase domain includes at least about 25-75, or more preferablyabout 45 amino acid residues, and has a bit score for the alignment ofthe sequence to the dehydrogenase domain of at least 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or higher. Theglucose-1-dehydrogenase domain has been assigned ProDom entry 77223. Toidentify the presence of a glucose-1-dehydrogenase domain in a DHDRprotein, and to make the determination that a protein of interest has aparticular profile, the amino acid sequence of the protein is searchedagainst a database of known protein domains (e.g., the ProDom database)using the default parameters (available online through the ProDomwebsite). A search was performed against the ProDom database resultingin the identification of a dehydrogenase domain in the amino acidsequence of human DHDR-4 (SEQ ID NO:11) at about residues 243-287 of SEQID NO:11. The results of the search are set forth in FIGS. 21A-21B.

[0087] In a preferred embodiment, the DHDR molecules of the inventioninclude at least one or more of the following domains: a transmembranedomain, a signal peptide domain, an aldehyde dehydrogenaseoxidoreductase domain, an aldehyde dehydrogenase family domain, a shortchain dehydrogenase domain, an oxidoreductase protein dehydrogenasedomain, a 3-beta hydroxysteroid dehydrogenase domain, a NAD-dependentepimerase/dehydratase domain, a short chain dehydrogenase/reductasedomain, a shikimate 5-dehydrogenase domain, a dehydrogenase domain, anda glucose-1-dehydrogenase domain.

[0088] Isolated proteins of the present invention, preferably DHDRproteins, have an amino acid sequence sufficiently identical to theamino acid sequence of SEQ ID NO:2, 5, 8, 11, or 15, or are encoded by anucleotide sequence sufficiently identical to SEQ ID NO:1, 3, 4, 6, 7,9, 10, 12, 14, or 16. As used herein, the term “sufficiently identical”refers to a first amino acid or nucleotide sequence which contains asufficient or minimum number of identical or equivalent (e.g., an aminoacid residue which has a similar side chain) amino acid residues ornucleotides to a second amino acid or nucleotide sequence such that thefirst and second amino acid or nucleotide sequences share commonstructural domains or motifs and/or a common functional activity. Forexample, amino acid or nucleotide sequences which share commonstructural domains have at least 30%, 40%, or 50% homology, preferably60% homology, more preferably 70%-80%, and even more preferably 90-95%homology across the amino acid sequences of the domains and contain atleast one and preferably two structural domains or motifs, are definedherein as sufficiently identical. Furthermore, amino acid or nucleotidesequences which share at least 30%, 40%, or 50%, preferably 60%, morepreferably 70-80%, or 90-95% homology and share a common functionalactivity are defined herein as sufficiently identical.

[0089] As used interchangeably herein, an “DHDR activity”, “biologicalactivity of DHDR” or “functional activity of DHDR”, refers to anactivity exerted by a DHDR protein, polypeptide or nucleic acid moleculeon a DHDR responsive cell or tissue, or on a DHDR protein substrate, asdetermined in vivo, or in vitro, according to standard techniques. Inone embodiment, a DHDR activity is a direct activity, such as anassociation with a DHDR-target molecule. As used herein, a “targetmolecule” or “binding partner” is a molecule with which a DHDR proteinbinds or interacts in nature, such that DHDR-mediated function isachieved. A DHDR target molecule can be a non-DHDR molecule or a DHDRprotein or polypeptide of the present invention (e.g., NAD+, NADP+, orother cofactor). In an exemplary embodiment, a DHDR target molecule is aDHDR ligand (e.g., an alcohol, an aldehyde, a lipid, or a steroid (e.g.,a glucocorticoid)). Alternatively, a DHDR activity is an indirectactivity, such as a cellular signaling activity mediated by interactionof the DHDR protein with a DHDR ligand. The biological activities ofDHDR are described herein. For example, the DHDR proteins of the presentinvention can have one or more of the following activities: 1) modulatemetabolism and catabolism of biochemical molecules necessary for energyproduction or storage, 2) modulate intra- or inter-cellular signaling,3) modulate metabolism or catabolism of metabolically importantbiomolecules (e.g., glucocorticoids), 4) modulate detoxification ofpotentially harmful compounds, 5) modulate viral infection (e.g., bymodulating viral gene expression), 6) act as a transcriptional cofactorfor viral gene activation, 7) modulate viral activity, and/or 8)modulate cellular proliferation.

[0090] Accordingly, another embodiment of the invention featuresisolated DHDR proteins and polypeptides having a DHDR activity. Otherpreferred proteins are DHDR proteins having one or more of the followingdomains: a transmembrane domain, a signal peptide domain, an aldehydedehydrogenase oxidoreductase domain, an aldehyde dehydrogenase familydomain, a short chain dehydrogenase domain, an oxidoreductase proteindehydrogenase domain, a 3-beta hydroxysteroid dehydrogenase domain, aNAD-dependent epimerase/dehydratase domain, a short chaindehydrogenase/reductase domain, a shikimate is 5-dehydrogenase domain, adehydrogenase domain, or a glucose-1-dehydrogenase domain and,preferably, a DHDR activity.

[0091] Additional preferred proteins have at least one transmembranedomain, and one or more of a signal peptide domain, an aldehydedehydrogenase oxidoreductase domain, an aldehyde dehydrogenase familydomain, a short chain dehydrogenase domain, an oxidoreductase proteindehydrogenase domain, a 3-beta hydroxysteroid dehydrogenase domain, aNAD-dependent epimerase/dehydratase domain, a short chaindehydrogenase/reductase domain, a shikimate 5-dehydrogenase domain, adehydrogenase domain, or a glucose-1-dehydrogenase domain., and are,preferably, encoded by a nucleic acid molecule having a nucleotidesequence which hybridizes under stringent hybridization conditions to acomplement of a nucleic acid molecule comprising the nucleotide sequenceof SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16.

[0092] The nucleotide sequence of the isolated human DHDR-1 cDNA and thepredicted amino acid sequence of the human DHDR-1 polypeptide are shownin FIGS. 1A-1D and in SEQ ID NOs:1 and 2, respectively. The nucleotidesequence of the isolated human DHDR-2 cDNA and the predicted amino acidsequence of the human DHDR-2 polypeptide are shown in FIGS. 6A-6B and inSEQ ID NOs:4 and 5, respectively. The nucleotide sequence of theisolated human DHDR-3 cDNA and the predicted amino acid sequence of thehuman DHDR-3 polypeptide are shown in FIGS. 11A-11B and in SEQ ID NOs:7and 8, respectively. The nucleotide sequence of the isolated humanDHDR-4 cDNA and the predicted amino acid sequence of the human DHDR-4polypeptide are shown in FIG. 16 and in SEQ ID NOs:10 and 11,respectively. The nucleotide sequence of the isolated mouse DHDR-2 andthe predicted amino acid sequence of the mouse DHDR-2 polypeptide areshown in FIGS. 31A and 31B, respectively, and in SEQ ID NOs:14 and 15,respectively. Plasmids containing the nucleotide sequence encoding humanDHDR-1, DHDR-2, DHDR-3, and DHDR-4, and mouse DHDR-2 were deposited withthe American Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110-2209, on ______. May 9, 2000, Mar. 22, 2001, and______, respectively, and assigned Accession Numbers ______, PTA-1845,______, ______, and ______, respectively. These deposits will bemaintained under the terms of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure. These deposits were made merely as a convenience for those ofskill in the art and are not an admission that deposits are requiredunder 35 U.S.C. §112.

[0093] The human DHDR-1 gene, which is approximately 2660 nucleotides inlength, encodes a protein having a molecular weight of approximately88.0 kD and which is approximately 802 amino acid residues in length.The human DHDR-2 gene, which is approximately 1379 nucleotides inlength, encodes a protein having a molecular weight of approximately34.2 kD and which is approximately 311 amino acid residues in length.The human DHDR-3 gene, which is approximately 1725 nucleotides inlength, encodes a protein having a molecular weight of approximately40.5 kD and which is approximately 369 amino acid residues in length.The human DHDR-4 gene, which is approximately 1209 nucleotides inlength, encodes a protein having a molecular weight of approximately35.4 kD and which is approximately 322 amino acid residues in length.The mouse DHDR-2 gene, which is approximately 1108 nucleotides inlength, encodes a protein having a molecular weight of approximately34.2 kD and which is approximately 311 amino acid residues in length.

[0094] Various aspects of the invention are described in further detailin the following subsections:

[0095] I. Isolated Nucleic Acid Molecules

[0096] One aspect of the invention pertains to isolated nucleic acidmolecules that encode DHDR proteins or biologically active portionsthereof, as well as nucleic acid fragments sufficient for use ashybridization probes to identify DHDR-encoding nucleic acid molecules(e.g., DHDR mRNA) and fragments for use as PCR primers for theamplification or mutation of DHDR nucleic acid molecules. As usedherein, the term “nucleic acid molecule” is intended to include DNAmolecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) andanalogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

[0097] The term “isolated nucleic acid molecule” includes nucleic acidmolecules which are separated from other nucleic acid molecules whichare present in the natural source of the nucleic acid. For example, withregards to genomic DNA, the term “isolated” includes nucleic acidmolecules which are separated from the chromosome with which the genomicDNA is naturally associated. Preferably, an “isolated” nucleic acid isfree of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated DHDR nucleic acid moleculecan contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1kb of nucleotide sequences which naturally flank the nucleic acidmolecule in genomic DNA of the cell from which the nucleic acid isderived. Moreover, an “isolated” nucleic acid molecule, such as a cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized.

[0098] A nucleic acid molecule of the present invention, e.g., a nucleicacid molecule having the nucleotide sequence of SEQ ID NO:1, 3, 4, 6, 7,9, 10, 12, 14, or 16, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number ______, PTA-1845,______, ______, or ______, or a portion thereof, can be isolated usingstandard molecular biology techniques and the sequence informationprovided herein. Using all or portion of the nucleic acid sequence ofSEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16, or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number ______, PTA-1845, ______, ______, or ______ as ahybridization probe, DHDR nucleic acid molecules can be isolated usingstandard hybridization and cloning techniques (e.g., as described inSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. . 2nd, ed., Cold Spring Harbor Laboratory, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0099] Moreover, a nucleic acid molecule encompassing all or a portionof SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16, or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number ______, PTA-1845, ______, ______, or ______ can beisolated by the polymerase chain reaction (PCR) using syntheticoligonucleotide primers designed based upon the sequence of SEQ ID NO:1,3, 4, 6, 7, 9, 10, 12, 14, or 16, or the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC as Accession Number ______,PTA-1845, ______, ______, or ______.

[0100] A nucleic acid of the invention can be amplified using cDNA, mRNAor, alternatively, genomic DNA as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to DHDR nucleotide sequencescan be prepared by standard synthetic techniques, e.g., using anautomated DNA synthesizer.

[0101] In a preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises the nucleotide sequence shown in SEQ ID NO:1, 3,4, 6, 7, 9, 10, 12, 14, or 16. This cDNA may comprise sequences encodingthe human DHDR-1 protein (i.e., “the coding region”, from nucleotides63-2468), as well as 5′ untranslated sequences (nucleotides 1-62) and 3′untranslated sequences (nucleotides 2469-2660) of SEQ ID NO: 1. ThiscDNA may comprise sequences encoding the human DHDR-2 protein (i.e.,“the coding region”, from nucleotides 331-1263), as well as 5′untranslated sequences (nucleotides 1-330) and 3′ untranslated sequences(nucleotides 1264-1379) of SEQ ID NO:4. This cDNA may comprise sequencesencoding the human DHDR-3 protein (i.e., “the coding region”, fromnucleotides 281-1387), as well as 5′ untranslated sequences (nucleotides1-280) and 3′ untranslated sequences (nucleotides 1388-1725) of SEQ IDNO:7. This cDNA may comprise sequences encoding the human DHDR-4 protein(i.e., “the coding region”, from nucleotides 61-1026), as well as 5′untranslated sequences (nucleotides 1-60) and 3′ untranslated sequences(nucleotides 1027-1209) of SEQ ID NO:10. This cDNA may comprisesequences encoding the mouse DHDR-2 protein (i.e., “the coding region”,from nucleotides 102-1034), as well as 5′ untranslated sequences(nucleotides 1-101) and 3′ untranslated sequences (nucleotides1035-1108) of SEQ ID NO:14. Alternatively, the nucleic acid molecule cancomprise only the coding region of SEQ ID NO:1 (e.g., nucleotides63-2468, corresponding to SEQ ID NO:3), only the coding region of SEQ IDNO:4 (e.g., nucleotides 331-1263, corresponding to SEQ ID NO:6), onlythe coding region of SEQ ID NO:7 (e.g., nucleotides 281-1387,corresponding to SEQ ID NO:9), only the coding region of SEQ ID NO:10(e.g., nucleotides 61-1026, corresponding to SEQ ID NO:12), or only thecoding region of SEQ ID NO:14 (e.g., nucleotides 102-1034, correspondingto SEQ ID NO:16).

[0102] In another preferred embodiment, an isolated nucleic acidmolecule of the invention comprises a nucleic acid molecule which is acomplement of the nucleotide sequence shown in SEQ ID NO:1, 3, 4, 6, 7,9, 10, 12, 14, or 16, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number ______, PTA-1845,______, ______ or ______, or a portion of any of these nucleotidesequences. A nucleic acid molecule which is complementary to thenucleotide sequence shown in SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or16, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number ______, PTA-1845, ______,______, or ______, is one which is sufficiently complementary to thenucleotide sequence shown in SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or16, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number ______, PTA-1845, ______,______, or ______ such that it can hybridize to the nucleotide sequenceshown in SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number ______, PTA-1845, ______, ______, or ______,respectively, thereby forming a stable duplex.

[0103] In still another preferred embodiment, an isolated nucleic acidmolecule of the present invention comprises a nucleotide sequence whichis at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 89%, 90%, 95%,96%, 97%, 98%, 99%, 99.1% 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,99.8%, 99.9%, 99.99% or more identical to the entire length of thenucleotide sequence shown in SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or16, or the entire length of the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number ______, PTA-1845,______, ______, or ______, or a portion of any of these nucleotidesequences.

[0104] Moreover, the nucleic acid molecule of the invention can compriseonly a portion of the nucleic acid sequence of SEQ ID NO:1, 3, 4, 6, 7,9, 10, 12, 14, or 16, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number ______, PTA-1845,______, ______, or ______, for example, a fragment which can be used asa probe or primer or a fragment encoding a portion of a DHDR protein,e.g., a biologically active portion of a DHDR protein. The nucleotidesequences determined from the cloning of the DHDR-1, DHDR-2, DHDR-3, andDHDR-4 genes allow for the generation of probes and primers designed foruse in identifying and/or cloning other DHDR family members, as well asDHDR homologues from other species. The probe/primer typically comprisessubstantially purified oligonucleotide. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12 or 15, preferably about 20 or25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75consecutive nucleotides of a sense sequence of SEQ ID NO:1, 3, 4, 6, 7,9, 10, 12, 14, or 16, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number ______, PTA-1845,______, ______, or of an anti-sense sequence of SEQ ID NO:1, 3, 4, 6, 7,9, 10, 12, 14, or 16, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number ______, PTA-1845,______, ______, or ______ or of a naturally occurring allelic variant ormutant of SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number ______, PTA-1845, ______, ______, or ______. In oneembodiment, a nucleic acid molecule of the present invention comprises anucleotide sequence which is greater than 50-100, 100-150, 150-200,200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600,600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000,1000-1050, 1050-1100, 1100-1150, 1150-1200, 1200-1250, 1250-1300,1300-1350, 1350-1400, 1400-1450, 1450-1500, 1500-1550,1550-1600,1600-1650, 1650-1700, 1700-1750, 1750-1800, 1800-1850,1850-1900, 1900-1950, 1950-2000 or more nucleotides in length andhybridizes under stringent hybridization conditions to a nucleic acidmolecule of SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number ______,PTA-1845, ______, ______, or ______.

[0105] Probes based on the DHDR nucleotide sequences can be used todetect transcripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto, e.g., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a diagnostic test kit for identifying cells ortissue which misexpress a DHDR protein, such as by measuring a level ofa DHDR-encoding nucleic acid in a sample of cells from a subject e.g.,detecting DHDR mRNA levels or determining whether a genomic DHDR genehas been mutated or deleted.

[0106] A nucleic acid fragment encoding a “biologically active portionof a DHDR protein” can be prepared by isolating a portion of thenucleotide sequence of SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16, orthe nucleotide sequence of the DNA insert of the plasmid deposited withATCC as Accession Number ______, PTA-1845, ______, ______, or ______which encodes a polypeptide having a DHDR biological activity (thebiological activities of the DHDR proteins are described herein),expressing the encoded portion of the DHDR protein (e.g., by recombinantexpression in vitro) and assessing the activity of the encoded portionof the DHDR protein.

[0107] The invention further encompasses nucleic acid molecules thatdiffer from the nucleotide sequence shown in SEQ ID NO:1, 3, 4, 6, 7, 9,10, 12, 14, or 16, or the nucleotide sequence of the DNA insert of theplasmid deposited with ATCC as Accession Number ______, PTA-1845,______, ______, or ______ due to degeneracy of the genetic code and thusencode the same DHDR proteins as those encoded by the nucleotidesequence shown in SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Nunber ______, PTA-1845, ______, ______, or ______. Inanother embodiment, an isolated nucleic acid molecule of the inventionhas a nucleotide sequence encoding a protein having an amino acidsequence shown in SEQ ID NO:2, 5, 8, 11, or 15.

[0108] In addition to the DHDR nucleotide sequences shown in SEQ IDNO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16, or the nucleotide sequence ofthe DNA insert of the plasmid deposited with ATCC as Accession Number______, PTA-1845, ______, ______, or ______, it will be appreciated bythose skilled in the art that DNA sequence polymorphisms that lead tochanges in the amino acid sequences of the DHDR proteins may existwithin a population (e.g., the human population). Such geneticpolymorphism in the DHDR genes may exist among individuals within apopulation due to natural allelic variation. As used herein, the terms“gene” and “recombinant gene” refer to nucleic acid molecules whichinclude an open reading frame encoding a DHDR protein, preferably amammalian DHDR protein, and can further include non-coding regulatorysequences, and introns.

[0109] Allelic variants of DHDR include both functional andnon-functional DHDR proteins. Functional allelic variants are naturallyoccurring amino acid sequence variants of the human DHDR protein thatmaintain the ability to bind a DHDR ligand or substrate and/or modulatecell proliferation and/or migration mechanisms. Functional allelicvariants will typically contain only conservative substitution of one ormore amino acids of SEQ ID NO:2, 5, 8, 11, or 15, or substitution,deletion or insertion of non-critical residues in non-critical regionsof the protein.

[0110] Non-functional allelic variants are naturally occurring aminoacid sequence variants of the human DHDR protein that do not have theability to either bind a DHDR ligand and/or modulate any of the DHDRactivities described herein. Non-functional allelic variants willtypically contain a non-conservative substitution, a deletion, orinsertion or premature truncation of the amino-acid sequence of SEQ IDNO:2, 5, 8, 11, or 15, or a substitution, insertion or deletion incritical residues or critical regions of the protein.

[0111] The present invention further provides non-human orthologues ofthe human DHDR proteins. Orthologues of the human DHDR protein areproteins that are isolated from non-human organisms and possess the sameDHDR ligand binding and/or modulation of membrane excitabilityactivities of the human DHDR protein. Orthologues of the human DHDRprotein can readily be identified as comprising an amino acid sequencethat is substantially identical to SEQ ID NO:2, 5, 8, or 11 (e.g., themouse DHDR-2 of SEQ ID NO:15).

[0112] Moreover, nucleic acid molecules encoding other DHDR familymembers and, thus, which have a nucleotide sequence which differs fromthe DHDR sequences of SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16, orthe nucleotide sequence of the DNA insert of the plasmid deposited withATCC as Accession Number ______, PTA-1845, ______, ______, or ______ areintended to be within the scope of the invention. For example, anotherDHDR cDNA can be identified based on the nucleotide sequence of human ormouse DHDR. Moreover, nucleic acid molecules encoding DHDR proteins fromdifferent species, and which, thus, have a nucleotide sequence whichdiffers from the DHDR sequences of SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12,14, or 16, or the nucleotide sequence of the DNA insert of the plasmiddeposited with-ATCC as Accession Number ______, PTA-1845, ______, or______ are intended to be within the scope of the invention. Forexample, a mouse DHDR cDNA can be identified based on the nucleotidesequence of a human DHDR.

[0113] Nucleic acid molecules corresponding to natural allelic variantsand homologues of the DHDR cDNAs of the invention can be isolated basedon their homology to the DHDR nucleic acids disclosed herein using thecDNAs disclosed herein, or a portion thereof, as a hybridization probeaccording to standard hybridization techniques under stringenthybridization conditions. Nucleic acid molecules corresponding tonatural allelic variants and homologues of the DHDR cDNAs of theinvention can further be isolated by mapping to the same chromosome orlocus as the DHDR gene.

[0114] Accordingly, in another embodiment, an isolated nucleic acidmolecule of the invention is at least 15, 20, 25, 30 or more nucleotidesin length and hybridizes under stringent conditions to the nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:1, 3, 4, 6, 7,9, 10, 12, 14, or 16, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number ______, PTA-1845,______, ______, or ______. In other embodiment, the nucleic acid is atleast 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400,400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800,800-850, 850-900, 900-950, 950-1000, 1000-1050, 1050-1100, 1100-1150,1150-1200, 1200-1250, 1250-1300, 1300-1350, 1350-1400, 1400-1450,1450-1500, 1500-1550, 1550-1600, 1600-1650, 1650-1700, 1700-1750,1750-1800, 1800-1850, 1850-1900, 1900-1950, 1950-2000 or morenucleotides in length.

[0115] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences that are significantly identical orhomologous to each other remain hybridized to each other. Preferably,the conditions are such that sequences at least about 70%, morepreferably at least about 80%, even more preferably at least about 85%or 90% identical to each other remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, Ausubel et al., eds.,John Wiley & Sons, Inc. (1995), sections 2, 4, and 6. Additionalstringent conditions can be found in Molecular Cloning: A LaboratoryManual, Sambrook et al., Cold Spring Harbor Press, Cold Spring Harbor,N.Y. (1989), chapters 7, 9, and 11. A preferred, non-limiting example ofstringent hybridization conditions includes hybridization in 4×sodiumchloride/sodium citrate (SSC), at about 65-70° C. (or alternativelyhybridization in 4×SSC plus 50% formamide at about 42-50° C.) followedby one or more washes in 1×SSC, at about 65-70° C. A preferred,non-limiting example of highly stringent hybridization conditionsincludes hybridization in 1×SSC, at about 65-70° C. (or alternativelyhybridization in 1×SSC plus 50% formamide at about 42-50° C.) followedby one or more washes in 0.3×SSC, at about 65-70° C. A preferred,non-limiting example of reduced stringency hybridization conditionsincludes hybridization in 4×SSC, at about 50-60° C. (or alternativelyhybridization in 6×SSC plus 50% formamide at about 40-45° C.) followedby one or more washes in 2×SSC, at about 50-60° C. Ranges intermediateto the above-recited values, e.g., at 65-70° C. or at 42-50° C. are alsointended to be encompassed by the present invention. SSPE (1×SSPE is0.15M NaCl, 10 mM NaH₂PO₄, and 1.25 mM EDTA, pH 7.4) can be substitutedfor SSC (1×SSC is 0.15M NaCl and 15mM sodium citrate) in thehybridization and wash buffers; washes are performed for 15 minutes eachafter hybridization is complete. The hybridization temperature forhybrids anticipated to be less than 50 base pairs in length should be5-10° C. less than the melting temperature (T_(m)) of the hybrid, whereT_(m) is determined according to the following equations. For hybridsless than 18 base pairs in length, T_(m)(° C.)=2(# of A+T bases)+4(# ofG+C bases). For hybrids between 18 and 49 base pairs in length, T_(m)(°C.)=81.5+16.6(log₁₀[Na⁺])+0.41(% G+C)−(600/N), where N is the number ofbases in the hybrid, and [Na⁺] is the concentration of sodium ions inthe hybridization buffer ([Na⁺] for 1×SSC=0.165 M). It will also berecognized by the skilled practitioner that additional reagents may beadded to hybridization and/or wash buffers to decrease non-specifichybridization of nucleic acid molecules to membranes, for example,nitrocellulose or nylon membranes, including but not limited to blockingagents (e.g., BSA or salmon or herring sperm carrier DNA), detergents(e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PVP and the like.When using nylon membranes, in particular, an additional preferred,non-limiting example of stringent hybridization conditions ishybridization in 0.25-0.5M NaH₂PO₄, 7% SDS at about 65° C., followed byone or more washes at 0.02M NaH₂PO₄, 1% SDS at 65° C. (see e.g., Churchand Gilbert (1984) Proc. Natl. Acad. Sci. USA 81:1991-1995), oralternatively 0.2×SSC, 1% SDS.

[0116] Preferably, an isolated nucleic acid molecule of the inventionthat hybridizes under stringent conditions-to the sequence of SEQ IDNO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16, and corresponds to anaturally-occurring nucleic acid molecule. As used herein, a“naturally-occurring” nucleic acid molecule refers to an RNA or DNAmolecule having a nucleotide sequence that occurs in nature (e.g.,encodes a natural protein).

[0117] In addition to naturally-occurring allelic variants of the DHDRsequences that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into thenucleotide sequences of SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14 or 16, orthe nucleotide sequence of the DNA insert of the plasmid deposited withATCC as Accession Number ______, PTA-1845, ______, ______, or ______,thereby leading to changes in the amino acid sequence of the encodedDHDR proteins, without altering the functional ability of the DHDRproteins. For example, nucleotide substitutions leading to amino acidsubstitutions at “non-essential” amino acid residues can be made in thesequence of SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number ______, PTA-1845, ______, ______, or ______. A“non-essential” amino acid residue is a residue that can be altered fromthe wild-type sequence of DHDR (e.g., the sequence of SEQ ID NO:2, 5, 8,11, or 15) without altering the biological activity, whereas an“essential” amino acid residue is required for biological activity. Forexample, amino acid residues that are conserved among the DHDR proteinsof the present invention, e.g., those present in a transmembrane domain,are predicted to be particularly unamenable to alteration. Furthermore,additional amino acid residues that are conserved between the DHDRproteins of the present invention and other members of the DHDR familyare not likely to be amenable to alteration.

[0118] Accordingly, another aspect of the invention pertains to nucleicacid molecules encoding DHDR proteins that contain changes in amino acidresidues that are not essential for activity. Such DHDR proteins differin amino acid sequence from SEQ ID NO:2, 5, 8, 11, or 15, yet retainbiological activity. In one embodiment, the isolated nucleic acidmolecule comprises a nucleotide sequence encoding a protein, wherein theprotein comprises an amino acid sequence at least about 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 89%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 92%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99% or moreidentical to SEQ ID NO:2, 5, 8, 11, or 15.

[0119] An isolated nucleic acid molecule encoding a DHDR proteinidentical to the protein of SEQ ID NO:2, 5, 8, 11, or 15 can be createdby introducing one or more nucleotide substitutions, additions ordeletions into the nucleotide sequence of SEQ ID NO:1, 3, 4, 6, 7, 9,10, 12, 14, or 16, or the nucleotide sequence of the DNA insert of theplasmid deposited with ATCC as Accession Number ______, PTA-1845,______, ______ or ______ such that one or more amino acid substitutions,additions or deletions are introduced into the encoded protein.Mutations can be introduced into SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14,or 16, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number ______, PTA-1845, ______,______, or ______ by standard techniques, such as site-directedmutagenesis and PCR-mediated mutagenesis. Preferably, conservative aminoacid substitutions are made at one or more predicted non-essential aminoacid residues. A “conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in a DHDRprotein is preferably replaced with another amino acid residue from thesame side chain family. Alternatively, in another embodiment, mutationscan be introduced randomly along all or part of a DHDR coding sequence,such as by saturation mutagenesis, and the resultant mutants can bescreened for DHDR biological activity to identify mutants that retainactivity. Following mutagenesis of SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12,14, or 16, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number ______, PTA-1845, ______,______, or ______, the encoded protein can be expressed recombinantlyand the activity of the protein can be determined.

[0120] In a preferred embodiment, a mutant DHDR protein can be assayedfor the ability to metabolize or catabolize biochemical moleculesnecessary for energy production or storage, permit intra- orinter-cellular signaling, metabolize or catabolize metabolicallyimportant biomolecules, and to detoxify potentially harmful compounds.

[0121] In addition to the nucleic acid molecules encoding DHDR proteinsdescribed above, another aspect of the invention pertains to isolatednucleic acid molecules which are antisense thereto. An “antisense”nucleic acid comprises a nucleotide sequence which is complementary to a“sense” nucleic acid encoding a protein, e.g., complementary to thecoding strand of a double-stranded cDNA molecule or complementary to anmRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bondto a sense nucleic acid. The antisense nucleic acid can be complementaryto an entire DHDR coding strand, or to only a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to a “codingregion” of the coding strand of a nucleotide sequence encoding a DHDR.The term “coding region” refers to the region of the nucleotide sequencecomprising codons which are translated into amino acid residues (e.g.,the coding region of human DHDR corresponds to SEQ ID NO:3, 6, 9 or 12,and the coding region of mouse DHDR corresponds to SEQ ID NO:16). Thecoding region may or may not include a stop codon. In anotherembodiment, the antisense nucleic acid molecule is antisense to a“noncoding region” of the coding strand of a nucleotide sequenceencoding DHDR. The term “noncoding region” refers to 5′ and 3′ sequenceswhich flank the coding region that are not translated into amino acids(i.e., also referred to as 5′ and 3′ untranslated regions).

[0122] Given the coding strand sequences encoding DHDR disclosed herein(e.g., SEQ ID NO:3, 6, 9, 12, or 16), antisense nucleic acids of theinvention can be designed according to the rules of Watson and Crickbase pairing. The antisense nucleic acid molecule can be complementaryto the entire coding region of DHDR mRNA, but more preferably is anoligonucleotide which is antisense to only a portion of the coding ornoncoding region of DHDR mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of DHDR mRNA. An antisense oligonucleotide canbe, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50nucleotides in length. An antisense nucleic acid of the invention can beconstructed using chemical synthesis and enzymatic ligation reactionsusing procedures known in the art. For example, an antisense nucleicacid (e.g., an antisense oligonucleotide) can be chemically synthesizedusing naturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. Examples of modifiednucleotides which can be used to generate the antisense nucleic acidinclude 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

[0123] The antisense nucleic acid molecules of the invention aretypically administered to a subject or generated in situ such that theyhybridize with or bind to cellular mRNA and/or genomic DNA encoding aDHDR protein to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of antisensenucleic acid molecules of the invention include direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For example, for systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

[0124] In yet another embodiment, the antisense nucleic acid molecule ofthe invention is an α-anomeric nucleic acid molecule. An α-anomericnucleic acid molecule forms specific double-stranded hybrids withcomplementary RNA in which, contrary to the usual β-units, the strandsrun parallel to each other (Gaultier et al. (1987) Nucleic Acids Res.15:6625-6641). The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

[0125] In still another embodiment, an antisense nucleic acid of theinvention is a ribozyme. Ribozymes are catalytic RNA molecules withribonuclease activity which are capable of cleaving a single-strandednucleic acid, such as an mRNA, to which they have a complementaryregion. Thus, ribozymes (e.g., hammerhead ribozymes (described inHaseloff and Gerlach (1988) Nature 334:585-591)) can be used tocatalytically cleave DHDR mRNA transcripts to thereby inhibittranslation of DHDR mRNA. A ribozyme having specificity for aDHDR-encoding nucleic acid can be designed based upon the nucleotidesequence of a DHDR cDNA disclosed herein (i.e., SEQ ID NO:1, 3, 4, 6, 7,9, 10, 12, 14, or 16, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number ______, PTA-1845,______, ______, or ______). For example, a derivative of a TetrahymenaL-19 IVS RNA can be constructed in which the nucleotide sequence of theactive site is complementary to the nucleotide sequence to be cleaved ina DHDR-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071;and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, DHDR mRNA can beused to select a catalytic RNA having a specific ribonuclease activityfrom a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W.(1993) Science 261:1411-1418.

[0126] Alternatively, DHDR gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of the DHDR(e.g., the DHDR promoter and/or enhancers; e.g., nucleotides 1-62 of SEQID NO:1, nucleotides 1-330 of SEQ ID NO:4, nucleotides 1-280 of SEQ IDNO:-7, nucleotides 1-60 of SEQ ID NO:10, or nucleotides 1-101 of SEQ IDNO:14) to form triple helical structures that prevent transcription ofthe DHDR gene in target cells. See generally, Helene, C. (1991)Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N. YAcad. Sci. 660:27-36; and Maher, L. J. (1992) Bioessays 14(12):807-15.

[0127] In yet another embodiment, the DHDR nucleic acid molecules of thepresent invention can be modified at the base moiety, sugar moiety orphosphate backbone to improve, e.g., the stability, hybridization, orsolubility of the molecule. For example, the deoxyribose phosphatebackbone of the nucleic acid molecules can be modified to generatepeptide nucleic acids (see Hyrup, B. and Nielsen, P. E. (1996) Bioorg.Med. Chem. 4(1):5-23). As used herein, the terms “peptide nucleic acids”or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which thedeoxyribose phosphate backbone is replaced by a pseudopeptide backboneand only the four natural nucleobases are retained. The neutral backboneof PNAs has been shown to allow for specific hybridization to DNA andRNA under conditions of low ionic strength. The synthesis of PNAoligomers can be performed using standard solid phase peptide synthesisprotocols as described in Hyrup and Nielsen (1996) supra andPerry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93:14670-675.

[0128] PNAs of DHDR nucleic acid molecules can be used in therapeuticand diagnostic applications. For example, PNAs can be used as antisenseor antigene agents for sequence-specific modulation of gene expressionby, for example, inducing transcription or translation arrest orinhibiting replication. PNAs of DHDR nucleic acid molecules can also beused in the analysis of single base pair mutations in a gene, (e.g., byPNA-directed PCR clamping); as ‘artificial restriction enzymes’ whenused in combination with other enzymes, (e.g., Hyrup and Nielsen (1996)supra)); or as probes or primers for DNA sequencing or hybridization(Hyrup and Nielsen (1996) supra; Perry-O'Keefe et al. (1996) supra).

[0129] In another embodiment, PNAs of DHDR can be modified, (e.g., toenhance their stability or cellular uptake), by attaching lipophilic orother helper groups to PNA, by the formation of PNA-DNA chimeras, or bythe use of liposomes or other techniques of drug delivery known in theart. For example, PNA-DNA chimeras of DHDR nucleic acid molecules can begenerated which may combine the advantageous properties of PNA and DNA.Such chimeras allow DNA recognition enzymes, (e.g., RNAse H and DNApolymerases), to interact with the DNA portion while the PNA portionwould provide high binding affinity and specificity. PNA-DNA chimerascan be linked using linkers of appropriate lengths selected in terms ofbase stacking, number of bonds between the nucleobases, and orientation(Hyrup and Nielsen (1996) supra). The synthesis of PNA-DNA chimeras canbe performed as described in Hyrup and Nielsen (1996) supra and Finn P.J. et al. (1996) Nucleic Acids Res. 24 (17):3357-63. For example, a DNAchain can be synthesized on a solid support using standardphosphoramidite coupling chemistry and modified nucleoside analogs,e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, canbe used as a between the PNA and the 5′ end of DNA (Mag, M. et al.(1989) Nucleic Acids Res. 17:5973-88). PNA monomers are then coupled ina stepwise manner to produce a chimeric molecule with a 5′ PNA segmentand a 3′ DNA segment (Finn P. J. et al. (1996) supra). Alternatively,chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNAsegment (Peterser, K. H. et al. (1975) Bioorganic Med. Chem. Lett.5:1119-11124).

[0130] In other embodiments, the oligonucleotide may include otherappended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier(see, e.g., PCT Publication No. W089/10134). In addition,oligonucleotides can be modified with hybridization-triggered cleavageagents (See, e.g., Krol et al. (1988) Biotechniques 6:958-976) orintercalating agents (see, e.g., Zon (1988) Pharm. Res. 5:539-549). Tothis end, the oligonucleotide may be conjugated to another molecule,(e.g., a peptide, hybridization triggered cross-linking agent, transportagent, or hybridization-triggered cleavage agent).

[0131] Alternatively, the expression characteristics of an endogenousDHDR gene within a cell line or microorganism may be modified byinserting a heterologous DNA regulatory element into the genome of astable cell line or cloned microorganism such that the insertedregulatory element is operatively linked with the endogenous DHDR gene.For example, an endogenous DHDR gene which is normally“transcriptionally silent”i.e., a DHDR gene which is normally notexpressed, or is expressed only at very low levels in a cell line ormicroorganism, may be activated by inserting a regulatory element whichis capable of promoting the expression of a normally expressed geneproduct in that cell line or microorganism. Alternatively, atranscriptionally silent, endogenous DHDR gene may be activated byinsertion of a promiscuous regulatory element that works across celltypes.

[0132] A heterologous regulatory element may be inserted into a stablecell line or cloned microorganism, such that it is operatively linkedwith an endogenous DHDR gene, using techniques, such as targetedhomologous recombination, which are well known to those of skill in theart, and described, e.g., in Chappel, U.S. Pat. No. 5,272,071; PCTpublication No. WO 91/06667, published May 16, 1991.

[0133] II. Isolated DHDR Proteins and Anti-DHDR Antibodies

[0134] One aspect of the invention pertains to isolated DHDR proteins,and biologically active portions thereof, as well aspolypeptide-fragments suitable for use as immunogens to raise anti-DHDRantibodies. In one embodiment, native DHDR proteins can be isolated fromcells or tissue sources by an appropriate purification scheme usingstandard protein purification techniques. In another embodiment, DHDRproteins are produced by recombinant DNA techniques. Alternative torecombinant expression, a DHDR protein or polypeptide can be synthesizedchemically using standard peptide synthesis techniques.

[0135] An “isolated” or “purified” protein or biologically activeportion thereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theDHDR protein is derived, or substantially free from chemical precursorsor other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations of DHDRprotein in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. In oneembodiment, the language “substantially free of cellular material”includes preparations of DHDR protein having less than about 30% (by dryweight) of non-DHDR protein (also referred to herein as a “contaminatingprotein”, more preferably less than about 20% of non-DHDR protein, stillmore preferably less than about 10% of non-DHDR protein, and mostpreferably less than about 5% non-DHDR protein. When the DHDR protein orbiologically active portion thereof is recombinantly produced, it isalso preferably substantially free of culture medium, i.e., culturemedium represents less than about 20%, more preferably less than about10%, and most preferably less than about 5% of the volume of the proteinpreparation.

[0136] The language “substantially free of chemical precursors or otherchemicals” includes preparations of DHDR protein in which the protein isseparated from chemical precursors or other chemicals which are involvedin the synthesis of the protein. In one embodiment, the language“substantially free of chemical precursors or other chemicals” includespreparations of DHDR protein having less than about 30% (by dry weight)of chemical precursors or non-DHDR chemicals, more preferably less thanabout 20% chemical precursors or non-DHDR chemicals, still morepreferably less than about 10% chemical precursors or non-DHDRchemicals, and most preferably less than about 5% chemical precursors ornon-DHDR chemicals.

[0137] As used herein, a “biologically active portion” of a DHDR proteinincludes a fragment of a DHDR protein which participates in aninteraction between a DHDR molecule and a non-DHDR molecule.Biologically active portions of a DHDR protein include peptidescomprising amino acid sequences sufficiently identical to or derivedfrom the amino acid sequence of the DHDR protein, e.g., the amino acidsequence shown in SEQ ID NO:2, 5, 8, 11, or 15, which include less aminoacids than the full length DHDR proteins, and exhibit at least oneactivity of a DHDR protein. Typically, biologically active portionscomprise a domain or motif with at least one activity of the DHDRprotein, e.g., modulating membrane excitability. A biologically activeportion of a DHDR protein can be a polypeptide which is, for example,25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800 or more amino acids in length. Biologicallyactive portions of a DHDR protein can be used as targets for developingagents which modulate a DHDR mediated activity, e.g., a proliferativeresponse.

[0138] In one embodiment, a biologically active portion of a DHDRprotein comprises at least one transmembrane domain. It is to beunderstood that a preferred biologically active portion of a DHDRprotein of the present invention may contain at least one transmembranedomain and one or more of the following domains: a signal peptidedomain, an aldehyde dehydrogenase oxidoreductase domain, an aldehydedehydrogenase family domain, a short chain dehydrogenase domain, anoxidoreductase protein dehydrogenase domain, a 3-beta hydroxysteroiddehydrogenase domain, a NAD-dependent epimerase/dehydratase domain, ashort chain dehydrogenase/reductase domain, a shikimate 5-dehydrogenasedomain, a dehydrogenase domain, or a glucose-1-dehydrogenase domain.Moreover, other biologically active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the functional activities of a native DHDRprotein.

[0139] In a preferred embodiment, the DHDR protein has an amino acidsequence shown in SEQ ID NO:2, 5, 8, 11, or 15. In other embodiments,the DHDR protein is substantially identical to SEQ ID NO:2, 5, 8, 11, or15, and retains the functional activity of the protein of SEQ ID NO:2,5, 8, 11, or 15, yet differs in amino acid sequence due to naturalallelic variation or mutagenesis, as described in detail in subsection Iabove. Accordingly, in another embodiment, the DHDR protein is a proteinwhich comprises an amino acid sequence at least about 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, 99.1%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% 99.9% 99.99% or moreidentical to SEQ ID NO:2, 5, 8, 11, or 15.

[0140] To determine the percent identity of two amino acid sequences orof two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-identical sequences can be disregarded for comparisonpurposes). In a preferred embodiment, the length of a reference sequencealigned for comparison purposes is at least 30% preferably at least 40%,more preferably at least 50%, even more preferably at least 60%, andeven more preferably at least 70%, 80%, or 90% of the length of thereference sequence (e.g., when aligning a second sequence to the DHDRamino acid sequence of SEQ ID NO:2, 5, 8, 11, or 15 having, e.g., 400amino acid residues, at least 120, preferably at least 160, morepreferably at least 200, even more preferably at least 240, and evenmore preferably at least 270, 320, 360 or more amino acid residues arealigned). The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

[0141] The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (J.Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporatedinto the GAP program in the GCG software package (available onlinethrough the website of the Genetics Computer Group), using either aBlosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10,8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet anotherpreferred embodiment, the percent identity between two nucleotidesequences is determined using the GAP program in the GCG softwarepackage (available online through the website of the Genetics ComputerGroup), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70,or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment,the percent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Meyers and W. Miller (Comput Appl.Biosci. 4:11-17 (1988)) which has been incorporated into the ALIGNprogram (version 2.0 or 2.0U), using a PAM 120 weight residue table, agap length penalty of 12 and a gap penalty of 4.

[0142] The nucleic acid and protein sequences of the present inventioncan further be used as a “query sequence” to perform a search againstpublic databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (1 990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to DHDR nucleic acid molecules of the invention. BLASTprotein searches can be performed with the XBLAST program, score=100,wordlength=3 to obtain amino acid sequences homologous to DHDR proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See the website of the NationalCenter for Biotechnology Information.

[0143] The invention also provides DHDR chimeric or fusion proteins. Asused herein, a DHDR “chimeric protein” or “fusion protein” comprises aDHDR polypeptide operatively linked to a non-DHDR polypeptide. An “DHDRpolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to a DHDR molecule, whereas a “non-DHDR polypeptide”refers to a polypeptide having an amino acid sequence corresponding to aprotein which is not substantially homologous to the DHDR protein, e.g.,a protein which is different from the DHDR protein and which is derivedfrom the same or a different organism. Within a DHDR fusion protein theDHDR polypeptide can correspond to all or a portion of a DHDR protein.In a preferred embodiment, a DHDR fusion protein comprises at least onebiologically active portion of a DHDR protein. In another preferredembodiment, a DHDR fusion protein comprises at least two biologicallyactive portions of a DHDR protein. Within the fusion protein, the term“operatively linked” is intended to indicate that the DHDR polypeptideand the non-DHDR polypeptide are fused in-frame to each other. Thenon-DHDR polypeptide can be fused to the N-terminus or C-terminus of theDHDR polypeptide.

[0144] For example, in one embodiment, the fusion protein is a GST-DHDRfusion protein in which the DHDR sequences are fused to the C-terminusof the GST sequences. Such fusion proteins can facilitate thepurification of recombinant DHDR.

[0145] In another embodiment, the fusion protein is a DHDR proteincontaining a heterologous signal sequence at its N-terminus. In certainhost cells (e.g., mammalian host cells), expression and/or secretion ofDHDR can be increased through use of a heterologous signal sequence.

[0146] The DHDR fusion proteins of the invention can be incorporatedinto pharmaceutical compositions and administered to a subject in vivo.The DHDR fusion proteins can be used to affect the bioavailability of aDHDR substrate. Use of DHDR fusion proteins may be usefultherapeutically for the treatment of disorders caused by, for example,(i) aberrant modification or mutation of a gene encoding a DHDR protein;(ii) mis-regulation of the DHDR gene; and (iii) aberrantpost-translational modification of a DHDR protein.

[0147] Moreover, the DHDR-fusion proteins of the invention can be usedas immunogens to produce anti-DHDR antibodies in a subject, to purifyDHDR ligands and in screening assays to identify molecules which inhibitthe interaction of DHDR with a DHDR substrate.

[0148] Preferably, a DHDR chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and reamplified to generatea chimeric gene sequence (see, for example, Current Protocols inMolecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). ADHDR-encoding nucleic acid can be cloned into such an expression vectorsuch that the fusion moiety is linked in-frame to the DHDR protein.

[0149] The present invention also pertains to variants of the DHDRproteins which function as either DHDR agonists (mimetics) or as DHDRantagonists. Variants of the DHDR proteins can be generated bymutagenesis, e.g., discrete point mutation or truncation of a DHDRprotein. An agonist of the DHDR proteins can retain substantially thesame, or a subset, of the biological activities of the naturallyoccurring form of a DHDR protein. An antagonist of a DHDR protein caninhibit one or more of the activities of the naturally occurring form ofthe DHDR protein by, for example, competitively modulating aDHDR-mediated activity of a DHDR protein. Thus, specific biologicaleffects can be elicited by treatment with a variant of limited function.In one embodiment, treatment of a subject with a variant having a subsetof the biological activities of the naturally occurring form of theprotein has fewer side effects in a subject relative to treatment withthe naturally occurring form of the DHDR protein.

[0150] In one embodiment, variants of a DHDR protein which function aseither DHDR agonists (mimetics) or as DHDR antagonists can be identifiedby screening combinatorial libraries of mutants, e.g., truncationmutants, of a DHDR protein for DHDR protein agonist or antagonistactivity. In one embodiment, a variegated library of DHDR variants isgenerated by combinatorial mutagenesis at the nucleic acid level and isencoded by a variegated gene library. A variegated library of DHDRvariants can be produced by, for example, enzymatically ligating amixture of synthetic oligonucleotides into gene sequences such that adegenerate set of potential DHDR sequences is expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display) containing the set of DHDR sequences therein.There are a variety of methods which can be used to produce libraries ofpotential DHDR variants from a degenerate oligonucleotide sequence.Chemical synthesis of a degenerate gene sequence can be performed in anautomatic DNA synthesizer, and the synthetic gene then ligated into anappropriate expression vector. Use of a degenerate set of genes allowsfor the provision, in one mixture, of all of the sequences encoding thedesired set of potential DHDR sequences. Methods for synthesizingdegenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem.53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983)Nucleic Acids Res. 11:477.

[0151] In addition, libraries of fragments of a DHDR protein codingsequence can be used to generate a variegated population of DHDRfragments for screening and subsequent selection of variants of a DHDRprotein. In one embodiment, a library of coding sequence fragments canbe generated by treating a double stranded PCR fragment of a DHDR codingsequence with a nuclease under conditions wherein nicking occurs onlyabout once per molecule, denaturing the double stranded DNA, renaturingthe DNA to form double stranded DNA which can include sense/antisensepairs from different nicked products, removing single stranded portionsfrom reformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the DHDR protein.

[0152] Several techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having aselected property. Such techniques are adaptable for rapid screening ofthe gene libraries generated by the combinatorial mutagenesis of DHDRproteins. The most widely used techniques, which are amenable to highthrough-put analysis, for screening large gene libraries typicallyinclude cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates isolation of the vectorencoding the gene whose product was detected. Recursive ensemblemutagenesis (REM), a new technique which enhances the frequency offunctional mutants in the libraries, can be used in combination with thescreening assays to identify DHDR variants (Arkin and Youvan (1992)Proc. Natl. Acad. Sci. USA 89:7811-7815; Delagrave et al. (1993) ProteinEng. 6(3):327-331).

[0153] In one embodiment, cell based assays can be exploited to analyzea variegated DHDR library. For example, a library of expression vectorscan be transfected into a cell line, e.g., a neuronal cell line, whichordinarily responds to a DHDR ligand in a particular DHDRligand-dependent manner. The transfected cells are then contacted with aDHDR ligand and the effect of expression of the mutant on, e.g.,membrane excitability of DHDR can be detected. Plasmid DNA can then berecovered from the cells which score for inhibition, or alternatively,potentiation of signaling by the DHDR ligand, and the individual clonesfurther characterized.

[0154] An isolated DHDR protein, or a portion or fragment thereof, canbe used as an immunogen to generate antibodies that bind DHDR usingstandard techniques for polyclonal and monoclonal antibody preparation.A full-length DHDR protein can be used or, alternatively, the inventionprovides antigenic peptide fragments of DHDR for use as immunogens. Theantigenic peptide of DHDR comprises at least 8 amino acid residues ofthe amino acid sequence shown in SEQ ID NO:2, 5, 8, 11, or 15 andencompasses an epitope of DHDR such that an antibody raised against thepeptide forms a specific immune complex with the DHDR protein.Preferably, the antigenic peptide comprises at least 10 amino acidresidues, more preferably at least 15 amino acid residues, even morepreferably at least 20 amino acid residues, and most preferably at least30 amino acid residues.

[0155] Preferred epitopes encompassed by the antigenic peptide areregions of DHDR that are located on the surface of the protein, e.g.,hydrophilic regions, as well as regions with high antigenicity (see, forexample, FIGS. 2, 7, 12, and 18).

[0156] A DHDR immunogen typically is used to prepare antibodies byimmunizing a suitable subject, (e.g., rabbit, goat, mouse or othermammal) with the immunogen. An appropriate immunogenic preparation cancontain, for example, recombinantly expressed DHDR protein or achemically synthesized DHDR polypeptide. The preparation can furtherinclude an adjuvant, such as Freund's complete or incomplete adjuvant,or similar immunostimulatory agent. Immunization of a suitable subjectwith an immunogenic DHDR preparation induces a polyclonal anti-DHDRantibody response.

[0157] Accordingly, another aspect of the invention pertains toanti-DHDR antibodies. The term “antibody” as used herein refers toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds (immunoreacts with) an antigen,such as a DHDR. Examples of immunologically active portions ofimmunoglobulin molecules include F(ab) and F(ab′)₂ fragments which canbe generated by treating the antibody with an enzyme such as pepsin. Theinvention provides polyclonal and monoclonal antibodies that bind DHDRmolecules. The term “monoclonal antibody” or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one species of an antigen binding sitecapable of immunoreacting with a particular epitope of DHDR. Amonoclonal antibody composition thus typically displays a single bindingaffinity for a particular DHDR protein with which it immunoreacts.

[0158] Polyclonal anti-DHDR antibodies can be prepared as describedabove by immunizing a suitable subject with a DHDR immunogen. Theanti-DHDR antibody titer in the immunized subject can be monitored overtime by standard techniques, such as with an enzyme linked immunosorbentassay (ELISA) using immobilized DHDR. If desired, the antibody moleculesdirected against DHDR can be isolated from the mammal (e.g., from theblood) and further purified by well known techniques, such as protein Achromatography to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the anti-DHDR antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975)Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol.127:539-46; Brown et al. (1980) J. Biol. Chem. 255:4980-83; Yeh et al.(1976) Proc. Natl. Acad. Sci. USA 76:2927-31; and Yeh et al. (1982) Int.J. Cancer 29:269-75), the more recent human B cell hybridoma technique(Kozbor et al. (1983) Immunol. Today 4:72), the EBV-hybridoma technique(Cole et al. (1985), Monclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96) or trioma techniques. The technology forproducing monoclonal antibody hybridomas is well known (see generally R.H. Kenneth, in Monoclonal Antibodies: A New Dimension In BiologicalAnalyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lerner(1981) Yale J. Biol. Med. 54:387-402; M. L. Gefter et al. (1977) SomaticCell Genet. 20 3:231-36). Briefly, an immortal cell line (typically amyeloma) is fused to lymphocytes (typically splenocytes) from a mammalimmunized with a DHDR immunogen as described above, and the culturesupernatants of the resulting hybridoma cells are screened to identify ahybridoma producing a monoclonal antibody that binds DHDR.

[0159] Any of the many well known protocols used for fusing lymphocytesand immortalized cell lines can be applied for the purpose of generatingan anti-DHDR monoclonal antibody (see, e.g., G. Galfre et al. (1977)Nature. 266:55052; Gefter et al. Somatic Cell Genet., cited supra;Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies,cited supra). Moreover, the ordinarily skilled worker will appreciatethat there are many variations of such methods which also would beuseful. Typically, the immortal cell line (e.g., a myeloma cell line) isderived from the same mammalian species as the lymphocytes. For example,murine hybridomas can be made by fusing lymphocytes from a mouseimmunized with an immunogenic preparation of the present invention withan immortalized mouse cell line. Preferred immortal cell lines are mousemyeloma cell lines that are sensitive to culture medium containinghypoxanthine, aminopterin and thymidine (“HAT medium”. Any of a numberof myeloma cell lines can be used as a fusion partner according tostandard techniques, e g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 orSp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCC.Typically, HAT-sensitive mouse myeloma cells are fused to mousesplenocytes using polyethylene glycol (“PEG”). Hybridoma cells resultingfrom the fusion are then selected using HAT medium, which kills unfusedand unproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindDHDR, e.g., using a standard ELISA assay.

[0160] Alternative to preparing monoclonal antibody-secretinghybridomas, a monoclonal anti-DHDR antibody can be identified andisolated by screening a recombinant combinatorial immunoglobulin library(e.g., an antibody phage display library) with DHDR to thereby isolateimmunoglobulin library members that bind DHDR. Kits for generating andscreening phage display libraries are commercially available (e.g., thePharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; andthe Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCTInternational Publication No. WO 92/18619; Dower et al. PCTInternational Publication No. WO 91/17271; Winter et al. PCTInternational Publication WO 92/20791; Markland et al. PCT InternationalPublication No. WO 92/15679; Breitling et al. PCT InternationalPublication WO 93/01288; McCafferty et al. PCT International PublicationNo. WO 92/01047; Garrard et al. PCT International Publication No. WO92/09690; Ladner et al. PCT International Publication No. WO 90/02809;Fuchs et al. (1991) Biotechnology (NY) 9:1369-1372; Hay et al. (1992)Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins etal. (1992) J. Mol. Biol. 226:889-896; Clarkson et al. (1991) Nature352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580;Garrard et al. (1991) Biotechnology (NY) 9:1373-1377; Hoogenboom et al.(1991) Nucleic Acids Res. 19:4133-4137; Barbas et al. (1991) Proc. Natl.Acad. Sci. USA 88:7978-7982; and McCafferty et al. (1990) Nature348:552-554.

[0161] Additionally, recombinant anti-DHDR antibodies, such as chimericand humanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in Robinson et al.International Application No. PCT/US86/02269; Akira, et al. EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison et al. European Patent Application 173,494; Neubergeret al. PCT International Publication No. WO 86/01533; Cabilly et al.U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987)Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol.139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218;Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al. (1985)Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al.(1986) BioTechniques 4:214; Winter U.S. Pat. 5,225,539; Jones et al.(1986) Nature 321:552-525; Verhoeyen et al. (1988) Science 239:1534; andBeidler et al. (1 988) J. Immunol. 141:4053-4060.

[0162] An anti-DHDR antibody (e.g., monoclonal antibody) can be used toisolate DHDR by standard techniques, such as affinity chromatography orimmunoprecipitation. An anti-DHDR antibody can facilitate thepurification of natural DHDR from cells and of recombinantly producedDHDR expressed in host cells. Moreover, an anti-DHDR antibody can beused to detect DHDR protein (e.g., in a cellular lysate or cellsupernatant) in order to evaluate the abundance and pattern ofexpression of the DHDR protein. Anti-DHDR antibodies can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling (i.e.,physically linking) the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

[0163] II. Recombinant Expression Vectors and Host Cells

[0164] Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a DHDR protein(or a portion thereof). As used herein, the term “vector” refers to anucleic acid molecule capable of transporting another nucleic acid towhich it has been linked. One type of vector is a “plasmid”, whichrefers to a circular double stranded DNA loop into which additional DNAsegments can be ligated. Another type of vector is a viral vector,wherein additional DNA segments can be ligated into the viral genome.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

[0165] The recombinant expression vectors of the invention comprise anucleic acid of the invention in a form suitable for expression of thenucleic acid in a host cell, which means that the recombinant expressionvectors include one or more regulatory sequences, selected on the basisof the host cells to be used for expression; which is operatively linkedto the nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory sequence(s)in a manner which allows for expression of the nucleotide sequence(e.g., in an in vitro transcription/translation system or in a host cellwhen the vector is introduced into the host cell). The term “regulatorysequence” is intended to include promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described, for example, in Goeddel (1990)Methods Enzymol. 185:3-7. Regulatory sequences include those whichdirect constitutive expression of a nucleotide sequence in many types ofhost cells and those which direct expression of the nucleotide sequenceonly in certain host cells (e.g., tissue-specific regulatory sequences).It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,and the like. The expression vectors of the invention can be introducedinto host cells to thereby produce proteins or peptides, includingfusion proteins or peptides, encoded by nucleic acids as describedherein (e.g., DHDR proteins, mutant forms of DHDR proteins, fusionproteins, and the like).

[0166] The recombinant expression vectors of the invention can bedesigned for expression of DHDR proteins in prokaryotic or eukaryoticcells. For example, DHDR proteins can be expressed in bacterial cellssuch as E. coli, insect cells (using baculovirus expression vectors)yeast cells or mammalian cells. Suitable host cells are discussedfurther in Goeddel (1990) supra. Alternatively, the recombinantexpression vector can be transcribed and translated in vitro, forexample using T7 promoter regulatory sequences and T7 polymerase.

[0167] Expression of proteins in prokaryotes is most often carried outin E. coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein.

[0168] Purified fusion proteins can be utilized in DHDR activity assays,(e.g., direct assays or competitive assays described in detail below),or to generate antibodies specific for DHDR proteins, for example. In apreferred embodiment, a DHDR fusion protein expressed in a retroviralexpression vector of the present invention can be utilized to infectbone marrow cells which are subsequently transplanted into irradiatedrecipients. The pathology of the subject recipient is then examinedafter sufficient time has passed (e.g., six (6) weeks).

[0169] Examples of suitable inducible non-fusion E. coli expressionvectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al. (1990) Methods Enzymol. 185:60-89). Target geneexpression from the pTrc vector relies on host RNA polymerasetranscription from a hybrid trp-lac fusion promoter. Target geneexpression from the pET 11d vector relies on transcription from a T7gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase(T7 gn1). This viral polymerase is supplied by host strains BL21(DE3) orHMS174(DE3) from a resident prophage harboring a T7 gn1 gene under thetranscriptional control of the lacUV 5 promoter.

[0170] One strategy to maximize recombinant protein expression in E.coli is to express the protein in a host bacteria with an impairedcapacity to proteolytically cleave the recombinant protein (Gottesman,S. (1990) Methods Enzymol. 185:119-128). Another strategy is to alterthe nucleic acid sequence of the nucleic acid to be inserted into anexpression vector so that the individual codons for each amino acid arethose preferentially utilized in E. coli (Wada et al., (1992) NucleicAcids Res. 20:2111-2118). Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques.

[0171] In another embodiment, the DHDR expression vector is a yeastexpression vector. Examples of vectors for expression in yeast S.cerevisiae include pYepSec1 (Baldari, et al., (1987) EMBO J. 6:229-234),pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz etal., (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego,Calif.), and picZ (Invitrogen Corp, San Diego, Calif.).

[0172] Alternatively, DHDR proteins can be expressed in insect cellsusing baculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., Sf9 cells)include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165)and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).

[0173] In yet another embodiment, a nucleic acid of the invention isexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987)Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

[0174] In another embodiment, the recombinant mammalian expressionvector is capable of directing expression of the nucleic acidpreferentially in a particular cell type (e.g., tissue-specificregulatory elements are used to express the nucleic acid).Tissue-specific regulatory elements are known in the art. Non-limitingexamples of suitable tissue-specific promoters include the albuminpromoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277),lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol.43:235-275), in particular promoters of T cell receptors (Winoto andBaltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al.(1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748),neuron-specific promoters (e.g., the neurofilament promoter; Byrne andRuddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477),pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, for examplethe murine hox promoters (Kessel and Gruss (1990) Science 249:374-379)and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev.3:537-546).

[0175] The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to DHDR mRNA. Regulatory sequences operatively linkedto a nucleic acid cloned in the antisense orientation can be chosenwhich direct the continuous expression of the antisense RNA molecule ina variety of cell types, for instance viral promoters and/or enhancers,or regulatory sequences can be chosen which direct constitutive, tissuespecific or cell type specific expression of antisense RNA. Theantisense expression vector can be in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense nucleic acids areproduced under the control of a high efficiency regulatory region, theactivity of which can be determined by the cell type into which thevector is introduced. For a discussion of the regulation of geneexpression using antisense genes see Weintraub, H. et al., Antisense RNAas a molecular tool for genetic analysis, Reviews—Trends in Genetics,Vol. 1(1) 1986.

[0176] Another aspect of the invention pertains to host cells into whicha DHDR nucleic acid molecule of the invention is introduced, e.g., aDHDR nucleic acid molecule within a recombinant expression vector or aDHDR nucleic acid molecule containing sequences which allow it tohomologously recombine into a specific site of the host cell's genome.The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

[0177] A host cell can be any prokaryotic or eukaryotic cell. Forexample, a DHDR protein can be expressed in bacterial cells such as E.coli, insect cells, yeast or mammalian cells (such as Chinese hamsterovary cells (CHO) or COS cells). Other suitable host cells are known tothose skilled in the art.

[0178] Vector DNA can be introduced into prokaryotic or eukaryotic cellsvia conventional transformation or transfection techniques. As usedherein, the terms “transformation” and “transfection” are intended torefer to a variety of art-recognized techniques for introducing foreignnucleic acid (e.g., DNA) into a host cell, including calcium phosphateor calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, or electroporation. Suitable methods fortransforming or transfecting host cells can be found in Sambrook, et al.(Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989), and other laboratory manuals.

[0179] For stable transfection of mammalian cells, it is known that,depending upon the expression vector and transfection technique used,only a small fraction of cells may integrate the foreign DNA into theirgenome. In order to identify and select these integrants, a gene thatencodes a selectable marker (e.g., resistance to antibiotics) isgenerally introduced into the host cells along with the gene ofinterest. Preferred selectable markers include those which conferresistance to drugs, such as G418, hygromycin and methotrexate. Nucleicacid encoding a selectable marker can be introduced into a host cell onthe same vector as that encoding a DHDR protein or can be introduced ona separate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

[0180] A host cell of the invention, such as a prokaryotic or eukaryotichost cell in culture, can be used to produce (i.e., express) a DHDRprotein. Accordingly, the invention further provides methods forproducing a DHDR protein using the host cells of the invention. In oneembodiment, the method comprises culturing the host cell of theinvention (into which a recombinant expression vector encoding a DHDRprotein has been introduced) in a suitable medium such that a DHDRprotein is produced. In another embodiment, the method further comprisesisolating a DHDR protein from the medium or the host cell.

[0181] The host cells of the invention can also be used to producenon-human transgenic animals. For example, in one embodiment, a hostcell of the invention is a fertilized oocyte or an embryonic stem cellinto which DHDR-coding sequences have been introduced. Such host cellscan then be used to create non-human transgenic animals in whichexogenous DHDR sequences have been introduced into their genome orhomologous recombinant animals in which endogenous DHDR sequences havebeen altered. Such animals are useful for studying the function and/oractivity of a DHDR and for identifying and/or evaluating modulators ofDHDR activity. As used herein, a “transgenic animal” is a non-humananimal, preferably a mammal, more preferably a rodent such as a rat ormouse, in which one or more of the cells of the animal includes atransgene. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, amphibians, and the like.A transgene is exogenous DNA which is integrated into the genome of acell from which a transgenic animal develops and which remains in thegenome of the mature animal, thereby directing the expression of anencoded gene product in one or more cell types or tissues of thetransgenic animal. As used herein, a “homologous recombinant animal” isa non-human animal, preferably a mammal, more preferably a mouse, inwhich an endogenous DHDR gene has been altered by homologousrecombination between the endogenous gene and an exogenous DNA moleculeintroduced into a cell of the animal, e.g., an embryonic cell of theanimal, prior to development of the animal.

[0182] A transgenic animal of the invention can be created byintroducing a DHDR-encoding nucleic acid into the male pronuclei of afertilized oocyte, e.g., by microinjection, retroviral infection, andallowing the oocyte to develop in a pseudopregnant female foster animal.The DHDR cDNA sequence of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ IDNO:9, or SEQ ID NO:14 can be introduced as a transgene into the genomeof a non-human animal. Alternatively, a nonhuman homologue of a humanDHDR gene, such as a mouse or rat DHDR gene, can be used as a transgene.Alternatively, a DHDR gene homologue, such as another DHDR familymember, can be isolated based on hybridization to the DHDR cDNAsequences of SEQ ID NO:1 or 3, SEQ ID NO:4 or 6, SEQ ID NO:7 or 9, SEQID NO:10 or 12, or SEQ ID NO:14 or 16, or the DNA insert of the plasmiddeposited with ATCC as Accession Number ______, PTA-1845, ______,______, or ______ (described further in subsection I above) and used asa transgene. Intronic sequences and polyadenylation signals can also beincluded in the transgene to increase the efficiency of expression ofthe transgene. A tissue-specific regulatory sequence(s) can be operablylinked to a DHDR transgene to direct expression of a DHDR protein toparticular cells. Methods for generating transgenic animals via embryomanipulation and microinjection, particularly animals such as mice, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of a DHDR transgene in its genome and/or expression of DHDRmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene encoding a DHDRprotein can further be bred to other transgenic animals carrying othertransgenes.

[0183] To create a homologous recombinant animal, a vector is preparedwhich contains at least a portion of a DHDR gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the DHDR gene. The DHDR gene can be a human gene(e.g., the cDNA of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9 or SEQ IDNO:12), but more preferably, is a non-human homologue of a human DHDRgene (e.g., SEQ ID NO:16, or a cDNA isolated by stringent hybridizationwith the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7 orSEQ ID NO:10). For example, a mouse DHDR gene can be used to construct ahomologous recombination nucleic acid molecule, e.g., a vector, suitablefor altering an endogenous DHDR gene in the mouse genome. In a preferredembodiment, the homologous recombination nucleic acid molecule isdesigned such that, upon homologous recombination, the endogenous DHDRgene is functionally disrupted (i e., no longer encodes a functionalprotein; also referred to as a “knock out” vector). Alternatively, thehomologous recombination nucleic acid molecule can be designed suchthat, upon homologous recombination, the endogenous DHDR gene is mutatedor otherwise altered but still encodes functional protein (e.g., theupstream regulatory region can be altered to thereby alter theexpression of the endogenous DHDR protein). In the homologousrecombination nucleic acid molecule, the altered portion of the DHDRgene is flanked at its 5′ and 3′ ends by additional nucleic acidsequence of the DHDR gene to allow for homologous recombination to occurbetween the exogenous DHDR gene carried by the homologous recombinationnucleic acid molecule and an endogenous DHDR gene in a cell, e.g., anembryonic stem cell. The additional flanking DHDR nucleic acid sequenceis of sufficient length for successful homologous recombination with theendogenous gene. Typically, several kilobases of flanking DNA (both atthe 5′ and 3′ ends) are included in the homologous recombination nucleicacid molecule (see, e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell51:503 for a description of homologous recombination vectors). Thehomologous recombination nucleic acid molecule is introduced into acell, e.g., an embryonic stem cell line (e.g., by electroporation) andcells in which the introduced DHDR gene has homologously recombined withthe endogenous DHDR gene are selected (see e.g., Li, E. et al. (1992)Cell 69:915). The selected cells can then injected into a blastocyst ofan animal (e.g., a mouse) to form aggregation chimeras (see e.g.,Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, E. J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term. Progeny harboringthe homologously recombined DNA in their germ cells call be used tobreed animals in which all cells of the animal contain the homologouslyrecombined DNA by germline transmission of the transgene. Methods forconstructing homologous recombination nucleic acid molecules, e.g.,vectors, or homologous recombinant animals are described further inBradley, A. (1991) Curr. Opin. Biotechnol. 2:823-829 and in PCTInternational Publication Nos. WO 90/11354 by Le Mouellec et al.; WO91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; and WO93/04169 by Berns et al.

[0184] In another embodiment, transgenic non-human animals can beproduced which contain selected systems which allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. (1992) Proc.Natl. Acad. Sci. USA 89:6232-6236. Another example of a recombinasesystem is the FLP recombinase system of Saccharomyces cerevisiae(O'Gorman et al. (1991) Science 251:1351-1355. If a cre/loxP recombinasesystem is used to regulate expression of the transgene, animalscontaining transgenes encoding both the Cre recombinase and a selectedprotein are required. Such animals can be provided through theconstruction of “double” transgenic animals, e.g., by mating twotransgenic animals, one containing a transgene encoding a selectedprotein and the other containing a transgene encoding a recombinase.

[0185] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut, I. et al.(1997) Nature 385:810-813 and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G₀ phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyte and then transferred to pseudopregnant femalefoster animal. The offspring borne of this female foster animal will bea clone of the animal from which the ell, e.g., the somatic cell, isisolated.

[0186] IV. Pharmaceutical Compositions

[0187] The DHDR nucleic acid molecules, fragments of DHDR proteins, andanti-DHDR antibodies (also referred to herein as “active compounds”) ofthe invention can be incorporated into pharmaceutical compositionssuitable for administration. Such compositions typically comprise thenucleic acid molecule, protein, or antibody and a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

[0188] A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

[0189] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

[0190] Sterile injectable solutions can be prepared by incorporating theactive compound (e.g., a fragment of a DHDR protein or an anti-DHDRantibody) in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

[0191] Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

[0192] For administration by inhalation, the compounds are delivered inthe form of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

[0193] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fuisidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

[0194] The compounds can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

[0195] In one embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

[0196] It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

[0197] Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Compounds which exhibit large therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

[0198] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

[0199] As defined herein, a therapeutically effective amount of proteinor polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The skilled artisan will appreciate that certainfactors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of a protein, polypeptide, orantibody can include a single treatment or, preferably, can include aseries of treatments.

[0200] In a preferred example, a subject is treated with antibody,protein, or polypeptide in the range of between about 0.1 to 20 mg/kgbody weight, one time per week for between about 1 to 10 weeks,preferably between 2 to 8 weeks, more preferably between about 3 to 7weeks, and even more preferably for about 4, 5, or 6 weeks. It will alsobe appreciated that the effective dosage of antibody, protein, orpolypeptide used for treatment may increase or decrease over the courseof a particular treatment. Changes in dosage may result and becomeapparent from the results of diagnostic assays as described herein.

[0201] The present invention encompasses agents which modulateexpression or activity. An agent may, for example, be a small molecule.For example, such small molecules include, but are not limited to,peptides, peptidomimetics, amino acids, amino acid analogs,polynucleotides, polynucleotide analogs, nucleotides, nucleotideanalogs, organic or inorganic compounds (i.e., including heteroorganicand organometallic compounds) having a molecular weight less than about10,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 5,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds. It is understood that appropriatedoses of small molecule agents depends upon a number of factors withinthe ken of the ordinarily skilled physician, veterinarian, orresearcher. The dose(s) of the small molecule will vary, for example,depending upon the identity, size, and condition of the subject orsample being treated, further depending upon the route by which thecomposition is to be administered, if applicable, and the effect whichthe practitioner desires the small molecule to have upon the nucleicacid or polypeptide of the invention.

[0202] Exemplary doses include milligram or microgram amounts of thesmall molecule per kilogram of subject or sample weight (e.g., about 1microgram per kilogram to about 500 milligrams per kilogram, about 100micrograms per kilogram to about 5 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram. It isfurthermore understood that appropriate doses of a small molecule dependupon the potency of the small molecule with respect to the expression oractivity to be modulated. Such appropriate doses may be determined usingthe assays described herein. When one or more of these small moleculesis to be administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian; or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

[0203] In certain embodiments of the invention, a modulator of DHDRactivity is administered in combination with other agents (e.g., a smallmolecule), or in conjunction with another, complementary treatmentregime. For example, in one embodiment, a modulator of DHDR activity isused to treat DHDR associated disorder. Accordingly, modulation of DHDRactivity may be used in conjunction with, for example, another agentused to treat the disorder (e.g., another agent used to treat a viral orcellular proliferation disorder).

[0204] Further, an antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

[0205] The conjugates of the invention can be used for modifying a givenbiological response, the drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor, alpha-interferon, beta-interferon, nerve growthfactor, platelet derived growth factor, tissue plasminogen activator;or, biological response modifiers such as, for example, lymphokines,interleukin-1 (“IL-1”, interleukin-2 (“IL-2”, interleukin-6 (“IL-6”,granulocyte macrophage colony stimulating factor (“GM-CSF”, granulocytecolony stimulating factor (“G-CSF”), or other growth factors.

[0206] Techniques for conjugating such therapeutic moiety to antibodiesare well known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol Rev. 62:119-58 (1982). Alternatively, an antibody can beconjugated to a second antibody to form an antibody heteroconjugate asdescribed by Segal in U.S. Pat. No. 4,676,980.

[0207] The nucleic acid molecules of the invention can be inserted intovectors and used as gene therapy vectors. Gene therapy vectors can bedelivered to a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

[0208] The pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

[0209] V. Uses and Methods of the Invention

[0210] The nucleic acid molecules, proteins, protein homologues, andantibodies described herein can be used in one or more of the followingmethods: a) screening assays; b) predictive medicine (e.g., diagnosticassays, prognostic assays, monitoring clinical trials, andpharmacogenetics); and c) methods of treatment (e.g., therapeutic andprophylactic). As described herein, a DHDR protein of the invention hasone or more of the following activities: 1) it modulates metabolism orcatabolism of biochemical molecules necessary for energy production orstorage, 2) it modulates intra- or inter-cellular signaling, 3) itmodulates metabolism or catabolism of metabolically importantbiomolecules (e.g., glucocorticoids), 4) it modulates detoxification ofpotentially harmful compounds, 5) it modulates viral infection (e.g., bymodulating viral gene expression), 6) it acts as a transcriptionalcofactor for viral gene activation, and/or 7) it modulates viralactivity, 8) it modulates cellular proliferation.

[0211] The isolated nucleic acid molecules of the invention can be used,for example, to express DHDR protein (e.g., via a recombinant expressionvector in a host cell in gene therapy applications), to detect DHDR mRNA(e.g., in a biological sample) or a genetic alteration in a DHDR gene,and to modulate DHDR activity, as described further below. The DHDRproteins can be used to treat disorders characterized by insufficient orexcessive production of a DHDR substrate or production of DHDRinhibitors. In addition, the DHDR proteins can be used to screen fornaturally occurring DHDR substrates, to screen for drugs or compoundswhich modulate DHDR activity, as well as to treat disorderscharacterized by insufficient or excessive production of DHDR protein orproduction of DHDR protein forms which have decreased, aberrant orunwanted activity compared to DHDR wild type protein (e.g.,dehydrogenase-associated disorders, such as CNS disorders; cardiacdisorders; muscular disorders; cellular growth, differentiation, ormigration disorders; neurological disorders; immune disorders; hormonaldisorders; and viral disorders. Moreover, the anti-DHDR antibodies ofthe invention can be used to detect and isolate DHDR proteins, regulatethe bioavailability of DHDR proteins, and modulate DHDR activity.

[0212] A. Screening Assays:

[0213] The invention provides a method (also referred to herein as a“screening assay”) for identifying modulators, e.g., candidate or testcompounds or agents (e.g., peptides, peptidomimetics, small molecules orother drugs) which bind to DHDR proteins, have a stimulatory orinhibitory effect on, for example, DHDR expression or DHDR activity, orhave a stimulatory or inhibitory effect on, for example, the expressionor activity of DHDR substrate.

[0214] In one embodiment, the invention provides assays for screeningcandidate or test compounds which are substrates of a DHDR protein orpolypeptide or biologically active portion thereof (e.g., aldehydes,alcohols, or steroids (e.g., glucocorticoids)). In another embodiment,the invention provides assays for screening candidate or test compoundswhich bind to or modulate the activity of a DHDR protein or polypeptideor biologically active portion thereof (e.g., cofactor or coenzymeanalogs, or inhibitory molecules). The test compounds of the presentinvention can be obtained using any of the numerous approaches incombinatorial library methods known in the art, including: biologicallibraries; spatially addressable parallel solid phase or solution phaselibraries; synthetic library methods requiring deconvolution; the‘one-bead one-compound’ library method; and synthetic library methodsusing affinity chromatography selection. The biological library approachis limited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Lam, K. S. (1997) Anticancer Drug Des. 12:145).

[0215] Examples of methods for the synthesis of molecular libraries canbe found in the art, for example in: DeWitt et al. (1993) Proc. Nat.Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

[0216] Libraries of compounds may be presented in solution (e.g.,Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria(Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409),plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) oron phage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990)Science 249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.).

[0217] In one embodiment, an assay is a cell-based assay in which a cellwhich expresses a DHDR protein or biologically active portion thereof iscontacted with a test compound and the ability of the test compound tomodulate DHDR activity is determined. Determining the ability of thetest compound to modulate DHDR activity can be accomplished bymonitoring, for example, the production of one or more specificmetabolites in a cell which expresses DHDR (see, e.g., Saada et al.(2000) Biochem. Biophys. Res. Commun. 269: 382-386). The cell, forexample, can be of mammalian origin, e.g., a liver cell, a neuronalcell, or a thymus cell.

[0218] The ability of the test compound to modulate DHDR binding to asubstrate (e.g., an alcohol, an aldehyde, or a steroid (e.g., aglucocorticoid)) or to bind to DHDR can also be determined. Determiningthe ability of the test compound to modulate DHDR binding to a substratecan be accomplished, for example, by coupling the DHDR substrate with aradioisotope or enzymatic label such that binding of the DHDR substrateto DHDR can be determined by detecting the labeled DHDR substrate in acomplex. Alternatively, DHDR could be coupled with a radioisotope orenzymatic label to monitor the ability of a test compound to modulateDHDR binding to a DHDR substrate in a complex. Determining the abilityof the test compound to bind DHDR can be accomplished, for example, bycoupling the compound with a radioisotope or enzymatic label such thatbinding of the compound to DHDR can be determined by detecting thelabeled DHDR compound in a complex. For example, compounds (e.g., DHDRsubstrates) can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directlyor indirectly, and the radioisotope detected by direct counting ofradioemission or by scintillation counting. Alternatively, compounds canbe enzymatically labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

[0219] It is also within the scope of this invention to determine theability of a compound (e.g., a DHDR substrate) to interact with DHDRwithout the labeling of any of the interactants. For example, amicrophysiometer can be used to detect the interaction of a compoundwith DHDR without the labeling of either the compound or the DHDR.McConnell, H. M. et al. (1992) Science 257:1906-1912. As used herein, a“microphysiometer” (e.g., Cytosensor) is an analytical instrument thatmeasures the rate at which a cell acidifies its environment using alight-addressable potentiometric sensor (LAPS). Changes in thisacidification rate can be used as an indicator of the interactionbetween a compound and DHDR.

[0220] In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a DHDR target molecule (e.g., a DHDRsubstrate) with a test compound and determining the ability of the testcompound to modulate (e.g., stimulate or inhibit) the activity of theDHDR target molecule. Determining the ability of the test compound tomodulate the activity of a DHDR target molecule can be accomplished, forexample, by determining the ability of the DHDR protein to bind to orinteract with the DHDR target molecule.

[0221] Determining the ability of the DHDR protein, or a biologicallyactive fragment thereof, to bind to or interact with a DHDR targetmolecule can be accomplished by one of the methods described above fordetermining direct binding. In a preferred embodiment, determining theability of the DHDR protein to bind to or interact with a DHDR targetmolecule can be accomplished by determining the activity of the targetmolecule. For example, the activity of the target molecule can bedetermined by detecting induction of a cellular response (i.e., changesin intracellular K⁺ levels or induction of viral gene expression),detecting catalytic/enzymatic activity of the target on an appropriatesubstrate, detecting the induction of a reporter gene (comprising atarget-responsive regulatory element operatively linked to a nucleicacid encoding a detectable marker, e.g., luciferase), or detecting atarget-regulated cellular response.

[0222] In yet another embodiment, an assay of the present invention is acell-free assay in which a DHDR protein or biologically active portionthereof is contacted with a test compound and the ability of the testcompound to bind to the DHDR protein or biologically active portionthereof is determined. Preferred biologically active portions of theDHDR proteins to be used in assays of the present invention includefragments which participate in interactions with non-DHDR molecules,e.g., fragments with high surface probability scores (see, for example,FIGS. 2, 7, 12, and 18). Binding of the test compound to the DHDRprotein can be determined either directly or indirectly as describedabove. In a preferred embodiment, the assay includes contacting the DHDRprotein or biologically active portion thereof with a known compoundwhich binds DHDR to form an assay mixture, contacting the assay mixturewith a test compound, and determining the ability of the test compoundto interact with a DHDR protein, wherein determining the ability of thetest compound to interact with a DHDR protein comprises determining theability of the test compound to preferentially bind to DHDR orbiologically active portion thereof as compared to the known compound.

[0223] In another embodiment, the assay is a cell-free assay in which aDHDR protein or biologically active portion thereof is contacted with atest compound and the ability of the test compound to modulate (e.g.,stimulate or inhibit) the activity of the DHDR protein or biologicallyactive portion thereof is determined. Determining the ability of thetest compound to modulate the activity of a DHDR protein can beaccomplished, for example, by determining the ability of the DHDRprotein to bind to a DHDR target molecule by one of the methodsdescribed above for determining direct binding. Determining the abilityof the DHDR protein to bind to a DHDR target molecule can also beaccomplished using a technology such as real-time BiomolecularInteraction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991)Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct.Biol. 5:699-705. As used herein, “BIA” is a technology for studyingbiospecific interactions in real time, without labeling any of theinteractants (e.g., BlAcore). Changes in the optical phenomenon ofsurface plasmon resonance (SPR) can be used as an indication ofreal-time reactions between biological molecules.

[0224] In an alternative embodiment, determining the ability of the testcompound to modulate the activity of a DHDR protein can be accomplishedby determining the ability of the DHDR protein to further modulate theactivity of a downstream effector of a DHDR target molecule. Forexample, the activity of the effector molecule on an appropriate targetcan be determined or the binding of the effector to an appropriatetarget can be determined as previously described.

[0225] In yet another embodiment, the cell-free assay involvescontacting a DHDR protein or biologically active portion thereof with aknown compound which binds the DHDR protein to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with the DHDR protein, whereindetermining the ability of the test compound to interact with the DHDRprotein comprises determining the ability of the DHDR protein topreferentially bind to or catalyze the transfer of a hydride moiety toor from the target substrate.

[0226] In more than one embodiment of the above assay methods of thepresent invention, it may be desirable to immobilize either DHDR or itstarget molecule to facilitate separation of complexed from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay. Binding of a test compound to a DHDR protein,or interaction of a DHDR protein with a target molecule in the presenceand absence of a candidate compound, can be accomplished in any vesselsuitable for containing the reactants. Examples of such vessels includemicrotitre plates, test tubes, and micro-centrifuge tubes. In oneembodiment, a fusion protein can be provided which adds a domain thatallows one or both of the proteins to be bound to a matrix. For example,glutathione-S-transferase/DHDR fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or DHDR protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level of DHDRbinding or activity determined using standard techniques.

[0227] Other techniques for immobilizing proteins on matrices can alsobe used in the screening assays of the invention. For example, either aDHDR protein or a DHDR target molecule can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated DHDR protein ortarget molecules can be prepared from biotin-NHS (N-hydroxysuccinimide)using techniques known in the art (e.g., biotinylation kit, PierceChemicals, Rockford, Ill.), and immobilized in the wells ofstreptavidin-coated 96 well plates (Pierce Chemical). Alternatively,antibodies reactive with DHDR protein or target molecules but which donot interfere with binding of the DHDR protein to its target moleculecan be derivatized to the wells of the plate, and unbound target or DHDRprotein trapped in the wells by antibody conjugation. Methods fordetecting such complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the DHDR protein or target molecule, as well asenzyme-linked assays which rely on detecting an enzymatic activityassociated with the DHDR protein or target molecule.

[0228] In another embodiment, modulators of DHDR expression areidentified in a method wherein a cell is contacted with a candidatecompound and the expression of DHDR mRNA or protein in the cell isdetermined. The level of expression of DHDR mRNA or protein in thepresence of the candidate compound is compared to the level ofexpression of DHDR mRNA or protein in the absence of the candidatecompound. The candidate compound can then be identified as a modulatorof DHDR expression based on this comparison. For example, whenexpression of DHDR mRNA or protein is greater (statisticallysignificantly greater) in the presence of the candidate compound than inits absence, the candidate compound is identified as a stimulator ofDHDR mRNA or protein expression. Alternatively, when expression of DHDRmRNA or protein is less (statistically significantly less) in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of DHDR mRNA or proteinexpression. The level of DHDR mRNA or protein expression in the cellscan be determined by methods described herein for detecting DHDR mRNA orprotein.

[0229] In yet another aspect of the invention, the DHDR proteins can beused as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO 94/10300), to identify other proteins, whichbind to or interact with DHDR (“DHDR-binding proteins” or “DHDR-6-bp”)and are involved in DHDR activity. Such DHDR-binding proteins are alsolikely to be involved in the propagation of signals by the DHDR proteinsor DHDR targets as, for example, downstream elements of a DHDR-mediatedsignaling pathway. Alternatively, such DHDR-binding proteins are likelyto be DHDR inhibitors.

[0230] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a DHDR protein isfused to a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, from alibrary of DNA sequences, that encodes an unidentified protein (“prey”or “sample”) is fused to a gene that codes for the activation domain ofthe known transcription factor. If the “bait” and the “prey” proteinsare able to interact, in vivo, forming a DHDR-dependent complex, theDNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., LacZ) which is operably linked to a transcriptionalregulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing thefunctional transcription factor can be isolated and used to obtain thecloned gene which encodes the protein which interacts with the DHDRprotein.

[0231] In another aspect, the invention pertains to a combination of twoor more of the assays described herein. For example, a modulating agentcan be identified using a cell-based or a cell free assay, and theability of the agent to modulate the activity of a DHDR protein can beconfirmed in vivo, e.g., in an animal such as an animal model forcellular transformation and/or tumorigenesis or an animal model forviral infection.

[0232] There are many animal models for viral infection known in theart. For example, a transgenic mouse model for hepatitis B virusinfection (HBV) (Guidotti, L. G. et al. (1995) J. Virol. 69:6158-6169)may be used. High-level viral gene expression is present in the liverand kidney tissues of these mice, and the hepatocytes of the micereplicate the virus at levels comparable to those in the infected liversof patients with chronic hepatitis.

[0233] Another mouse model for HBV infection that may be used includesthe mouse model made by transplanting primary human hepatocytes intomice in a matrix under the kidney capsule along with administration ofan agonistic antibody against c-Met (Ohashi, K. et al. (2000) Nat. Med.6:327-331). These mice are susceptible to HBV infection. Additionally,they are susceptible to super-infection with hepatitis delta virus(HDV).

[0234] Other mouse models for HBV infection that may be used include themice described in Babinet, C. et al. (1985) Science 230:1160-3; Lee,T.-H. et al. (1990) J. Virol. 64:5939-5947; Madden, C. R. et al. (2000)J. Virol. 74:5266-5272; Brown, J. J. et al. (2000) Hepatology31:173-181; Larkin, J. (1999) Nat. Med. 5:907-912; and Araki, K. et al.(1989) Proc. Natl. Acad. Sci. USA 86:207-11.

[0235] Chronic HBV infection is a major risk factor for hepatocellularcarcinoma (Beasley, R. P. (1988) Cancer 61:1942-1956; Slagle, B. et al.(1994) In Viruses and cancer, Minson, A. et al., eds., University ofCambridge, Cambridge, England 51:149-171), and mice transgenic for theHBV X gene have increased sensitivity to hepatocarcinogens (Slagle, B.L. et al. (1996) Mol. Carcinog. 15:261-269). The double transgenic mousestrain described in Madden et al. (supra) can be used to study theeffects of test compounds identified by the screening methods of theinvention in modulating HBV X-mediated hepatocarcinogen sensitivity. Forexample, the mice can be treated with a hepatocarcinogen and a testcompound, and the effect of the test compound on thehepatocarcinogen-mediated mutation rate of the host DNA can be assayedby functional analysis of a bacteriophage lambda transgene. Briefly, DNAisolated from the livers of such treated mice can be packaged intolambda phage particles and used to infect E. coli bacteria. Mutationrates of the lambda particles (methods for determination of which areknown in the art) are directly related to the HBV X-mediated host DNAmutation rates in response to the hepatorcarcinogen in the treated mice.

[0236] Other mouse models for HBV infection and HBV immunity that may beused include those made by transplanting human peripheral bloodmononuclear cells (PBMC) from chronic HBV carriers and HBV-immunizeddonors, respectively, into lethally-irradiated Balb/c mice (Böcher, W.O. et al. (2000) Hepatology 31 :480-487; Ilan, E. et al. (1999)Hepatology 29:553-562). Such human/mouse radiation chimeras, calledTrimera mice, may be used to study the effects of test compoundsidentified by the screening methods of the invention on human antibodyand T cell responses to HBV infection in vivo (Marcus, H. et al. (1995)Blood 86:398-406; Reisner, Y. et al. (1998) Trends Biotechnol.16:242-246; Segall, H. et al. (1996) Blood 88:721-730; Böcher, W. O. etal. (1999) Immunology 96:634-641).

[0237] The effects of a modulating agent on HBV infection can also bestudied in other hepadnavirus animal models: the woodchuck hepatitisvirus (WHV) model (Korba, B. E. et al. (2000) Hepatology 31:1165-1175;Cote, P. J. et al. (2000) Hepatology 31:190-200), the duck hepatitis Bvirus (DHBV) model (Le Guerhier, F. et al. (2000) Antimicrob. AgentsChemother. 44:111-122; Vickery, K. et al. (1999) J. Med Virol.58:19-25), and the chimpanzee and ground and tree squirrel models(Caselmann, W. H. (1994) Antiviral Res. 24:121-129).

[0238] While an animal model for hepatitis C virus (HCV) infection thatadequately reproduces the characteristics of HCV infection in humansdoes not yet exist, there is an HCV Trimera mouse model (Dekel, B. etal. (1995) J. Infect. Dis. 172:25-30), and there are some mouse strainsthat are transgenic for certain HCV proteins, and thus, may be usefulfor testing compounds that can modulate DHDR activity in vivo(Pasquinelli, C. et al. (1997) Hepatology 25:719-727).

[0239] Other animal models for viral infection are also known in the artand may be used in the screening assays of the present invention. Forexample, there are many animal models for Epstein-Barr virus (EBV)associated lymphoproliferative disease. Such models have been made inrabbits, common marmosets (Callithrix jacchus), cottontop tamarins(Saguinus oedipus oedipus), rhesus monkeys, and the severe combinedimmunodeficient (SLID) mouse (Johannessen, I. and Crawford, D. H. (1999)Rev. Med. Virol. 9:263-77; Hayashi, K. and Akagi, T. (2000) Path.International 50:85-97). The mouse γ-herpesvirus 68 infection model(Speck, S. H. and Virgin, H. W. (1999) Curr. Opin. Microbiol. 2:403-9;Virgin, H. W. and Speck, S. H. (1999) Curr. Opin. Immunol. 11:371-379)and the cotton rat model for measles virus infection (Niewiesk, S.(1999) Immunol. Lett. 65:47-50) present other examples of animal modelsthat may be used in the methods of the invention. Macaques infected withlive attenuated simian immunodeficiency virus (SIV) (Geretti, A. M.(1999) Rev. Med. Virol. 9:57-67; Almond, N. and Stott, J. (1999) ImmunolLett. 66:167-170) as well as the chimpanzee HIV model (Murthy, K. K. etal. (1998) AIDS Res. Hum. Retroviruses 14 Suppl 3: S271-6) can be usedas models for human immunodeficiency virus (HIV) infection.

[0240] Other examples of animal models that may be used in the methodsof the invention include the transgenic mouse model for an AIDS-likedisease (Renkema, H. G. and Saksela, K. (2000) Front. Biosci. 5:D268-83); the chicken model for lymphoma-inducing herpesviruses (Schat,K. A. and Xing, Z. (2000) Dev. Comp. Immunol. 24:201-21); and the mousemodel of cytomegalovirus infection (Sweet, C. (1999) FEMS Microbiol Rev.23:457-82).

[0241] In another embodiment of the invention, the ability of the agentto modulate the activity of a DHDR protein can be tested in an animalsuch as an animal model for a cellular proliferation disorder, e.g.,tumorigenesis. Animal based models for studying tumorigenesis in vivoare well known in the art (reviewed in Animal Models of CancerPredisposition Syndromes, Hiai, H. and Hino, O. (eds.) 1999, Progress inExperimental Tumor Research, Vol. 35; Clarke, A. R. (2000)Carcinogenesis 21:435-41) and include, for example, carcinogen-inducedtumors (Rithidech, K. et al. (1999) Mutat. Res. 428:33-39; Miller, M. L.et al. (2000) Environ. Mol. Mutagen. 35:319-327), injection and/ortransplantation of tumor cells into an animal, as well as animalsbearing mutations in growth regulatory genes, for example, oncogenes(e.g., ras) (Arbeit, J. M. et al. (1993) Am. J. Pathol. 142:1187-1197;Sinn, E. et al. (1987) Cell 49:465-475; Thorgeirsson, S S et al. ToxicolLett (2000) 112-113:553-555and tumor suppressor genes (e.g., p53)(Vooijs, M. et al. (1999) Oncogene 18:5293-5303; Clark A. R. (1995)Cancer Metast. Rev. 14:125-148; Kumar, T. R. et al. (1995) J. Intern.Med. 238:233-238; Donehower, L. A. et al. (1992) Nature 356215-221).Furthermore, experimental model systems are available for the study of,for example, ovarian cancer (Hamilton, T. C. et al. (1984) Semin. Oncol11:285-298; Rahman, N. A. et al. (1998) Mol. Cell. Endocrinol.145:167-174; Bearner, W. G. et al. (1998) Toxicol. Pathol. 26:704-710),gastric cancer (Thompson, J. et al. (2000) Int. J. Cancer 86:863-869;Fodde, R. et al. (1999) Cytogenet. Cell Genet. 86:105-111), breastcancer (Li, M. et al. (2000) Oncogene 19:1010-1019; Green, J. E. et al.(2000) Oncogene 19:1020-1027), melanoma (Satyamoorthy, K. et al. (1999)Cancer Metast. Rev. 18:401-405), and prostate cancer (Shirai, T. et al.(2000) Mutat. Res. 462:219-226; Bostwick, D. G. et al. (2000) Prostate43:286-294).

[0242] This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model, as described above. For example, an agentidentified as described herein (e.g., a DHDR modulating agent, anantisense DHDR nucleic acid molecule, a DHDR-specific antibody, or aDHDR-binding partner) can be used in an animal model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal model to determine the mechanism of action of such an agent.Furthermore, this invention pertains to uses of novel agents identifiedby the above-described screening assays for treatments as describedherein.

[0243] B. Detection Assays

[0244] Portions or fragments of the cDNA sequences identified herein(and the corresponding complete gene sequences) can be used in numerousways as polynucleotide reagents. For example, these sequences can beused to: (i) map their respective genes on a chromosome; and, thus,locate gene regions associated with genetic disease; (ii) identify anindividual from a minute biological sample (tissue typing); and (iii)aid in forensic identification of a biological sample. Theseapplications are described in the subsections below.

[0245] 1. Chromosome Mapping

[0246] Once the sequence (or a portion of the sequence) of a gene hasbeen isolated, this sequence can be used to map the location of the geneon a chromosome. This process is called chromosome mapping. Accordingly,portions or fragments of the DHDR nucleotide sequences, describedherein, can be used to map the location of the DHDR genes on achromosome. The mapping of the DHDR sequences to chromosomes is animportant first step in correlating these sequences with genesassociated with disease.

[0247] Briefly, DHDR genes can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp in length) from the DHDR nucleotidesequences. Computer analysis of the DHDR sequences can be used topredict primers that do not span more than one exon in the genomic DNA,thus complicating the amplification process. These primers can then beused for PCR screening of somatic cell hybrids containing individualhuman chromosomes. Only those hybrids containing the human genecorresponding to the DHDR sequences will yield an amplified fragment.

[0248] Somatic cell hybrids are prepared by fusing somatic cells fromdifferent mammals (e.g., human and mouse cells). As hybrids of human andmouse cells grow and divide, they gradually lose human chromosomes inrandom order, but retain the mouse chromosomes. By using media in whichmouse cells cannot grow, because they lack a particular enzyme, buthuman cells can, the one human chromosome that contains the geneencoding the needed enzyme, will be retained. By using various media,panels of hybrid cell lines can be established. Each cell line in apanel contains either a single human chromosome or a small number ofhuman chromosomes, and a full set of mouse chromosomes, allowing easymapping of individual genes to specific human chromosomes (D'EustachioP. et al. (1983) Science 220:919-924). Somatic cell hybrids containingonly fragments of human chromosomes can also be produced by using humanchromosomes with translocations and deletions.

[0249] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular sequence to a particular chromosome. Three ormore sequences can be assigned per day using a single thermal cycler.Using the DHDR nucleotide sequences to design oligonucleotide primers,sublocalization can be achieved with panels of fragments from specificchromosomes. Other mapping strategies which can similarly be used to mapa DHDR sequence to its chromosome include in situ hybridization(described in Fan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA87:6223-27), pre-screening with labeled flow-sorted chromosomes, andpre-selection by hybridization to chromosome specific cDNA libraries.

[0250] Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. Chromosome spreads can be made usingcells whose division has been blocked in metaphase by a chemical such ascolcemid that disrupts the mitotic spindle. The chromosomes can betreated briefly with trypsin, and then stained with Giemsa. A pattern oflight and dark bands develops on each chromosome, so that thechromosomes can be identified individually. The FISH technique can beused with a DNA sequence as short as 500 or 600 bases. However, cloneslarger than 1,000 bases have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Preferably 1,000 bases, and more preferably 2,000 bases willsuffice to get good results at a reasonable amount of time. For a reviewof this technique, see Verma et al., Human Chromosomes: A Manual ofBasic Techniques (Pergamon Press, New York 1988).

[0251] Reagents for chromosome mapping can be used individually to marka single chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

[0252] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data (such data are found, for example, inV. McKusick, Mendelian Inheritance in Man, available on-line throughJohns Hopkins University Welch Medical Library). The relationshipbetween a gene and a disease, mapped to the same chromosomal region, canthen be identified through linkage analysis (co-inheritance ofphysically adjacent genes), described in, for example, Egeland, J. etal. (1987) Nature, 325:783-787.

[0253] Moreover, differences in the DNA sequences between individualsaffected and unaffected with a disease associated with the DHDR gene canbe determined. If a mutation is observed in some or all of the affectedindividuals but not in any unaffected individuals, then the mutation islikely to be the causative agent of the particular disease. Comparisonof affected and unaffected individuals generally involves first lookingfor structural alterations in the chromosomes, such as deletions ortranslocations that are visible from chromosome spreads or detectableusing PCR based on that DNA sequence. Ultimately, complete sequencing ofgenes from several individuals can be performed to confirm the presenceof a mutation and to distinguish mutations from polymorphisms.

[0254] 2. Tissue Typing

[0255] The DHDR sequences of the present invention can also be used toidentify individuals from minute biological samples. The United Statesmilitary, for example, is considering the use of restriction fragmentlength polymorphism (RFLP) for identification of its personnel. In thistechnique, an individual's genomic DNA is digested with one or morerestriction enzymes, and probed on a Southern blot to yield unique bandsfor identification. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The sequences of the presentinvention are useful as additional DNA markers for RFLP (described inU.S. Pat. No. 5,272,057).

[0256] Furthermore, the sequences of the present invention can be usedto provide an alternative technique which determines the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. Thus, the DHDR nucleotide sequences described herein can be usedto prepare two PCR primers from the 5′ and 3′ ends of the sequences.These primers can then be used to amplify an individual's DNA andsubsequently sequence it.

[0257] Panels of corresponding DNA sequences from individuals, preparedin this manner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The DHDR nucleotide sequences of the invention uniquely representportions of the human genome. Allelic variation occurs to some degree inthe coding regions of these sequences, and to a greater degree in thenoncoding regions. It is estimated that allelic variation betweenindividual humans occurs with a frequency of about once per each 500bases. Each of the sequences described herein can, to some degree, beused as a standard against which DNA from an individual can be comparedfor identification purposes. Because greater numbers of polymorphismsoccur in the noncoding regions, fewer sequences are necessary todifferentiate individuals. The noncoding sequences of SEQ ID NO:1, SEQID NO:4, SEQ ID NO:7, or SEQ ID NO:10 can comfortably provide positiveindividual identification with a panel of perhaps 10 to 1,000 primerswhich each yield a noncoding amplified sequence of 100 bases. Ifpredicted coding sequences, such as those in SEQ ID NO:3, 6, 9, or 12are used, a more appropriate number of primers for positive individualidentification would be 500-2,000.

[0258] If a panel of reagents from DHDR nucleotide sequences describedherein is used to generate a unique identification database for anindividual, those same reagents can later be used to identify tissuefrom that individual. Using the unique identification database, positiveidentification of the individual, living or dead, can be made fromextremely small tissue samples.

[0259] 3. Use of DHDR Sequences in Forensic Biology

[0260] DNA-based identification techniques can also be used in forensicbiology. Forensic biology is a scientific field employing genetic typingof biological evidence found at a crime scene as a means for positivelyidentifying, for example, a perpetrator of a crime. To make such anidentification, PCR technology can be used to amplify DNA sequencestaken from very small biological samples such as tissues, e.g., hair orskin, or body fluids, e.g., blood, saliva, or semen found at a crimescene. The amplified sequence can then be compared to a standard,thereby allowing identification of the origin of the biological sample.

[0261] The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e., another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7or SEQ ID NO:10 are particularly appropriate for this use as greaternumbers of polymorphisms occur in the noncoding regions, making iteasier to differentiate individuals using this technique. Examples ofpolynucleotide reagents include the DHDR nucleotide sequences orportions thereof, e.g., fragments derived from the noncoding regions ofSEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7 or SEQ ID NO:10 having a length ofat least 20 bases, preferably at least 30 bases.

[0262] The DHDR nucleotide sequences described herein can further beused to provide polynucleotide reagents, e.g., labeled or labelableprobes which can be used in, for example, an in situ hybridizationtechnique, to identify a specific tissue, e.g., thymus or brain tissue.This can be very useful in cases where a forensic pathologist ispresented with a tissue of unknown origin. Panels of such DHDR probescan be used to identify tissue by species and/or by organ type.

[0263] In a similar fashion, these reagents, e.g., DHDR primers orprobes can be used to screen tissue culture for contamination (i.e.,screen for the presence of a mixture of different types of cells in aculture).

[0264] C. Predictive Medicine:

[0265] The present invention also pertains to the field of predictivemedicine in which diagnostic assays, prognostic assays, and monitoringclinical trials are used for prognostic (predictive) purposes to therebytreat an individual prophylactically. Accordingly, one aspect of thepresent invention relates to diagnostic assays for determining DHDRprotein and/or nucleic acid expression as well as DHDR activity, in thecontext of a biological sample (e.g., blood, serum, cells, tissue) tothereby determine whether an individual is afflicted with a disease ordisorder, or is at risk of developing a disorder, associated withaberrant or unwanted DHDR expression or activity. The invention alsoprovides for prognostic (or predictive) assays for determining whetheran individual is at risk of developing a disorder associated with DHDRprotein, nucleic acid expression or activity. For example, mutations ina DHDR gene can be assayed in a biological sample. Such assays can beused for prognostic or predictive purpose to thereby prophylacticallytreat an individual prior to the onset of a disorder characterized by orassociated with DHDR protein, nucleic acid expression or activity.

[0266] Another aspect of the invention pertains to monitoring theinfluence of agents (e.g., drugs, compounds) on the expression oractivity of DHDR in clinical trials.

[0267] These and other agents are described in further detail in thefollowing sections.

[0268] 1. Diagnostic Assays

[0269] An exemplary method for detecting the presence or absence of DHDRprotein or nucleic acid in a biological sample involves obtaining abiological sample from a test subject and contacting the biologicalsample with a compound or an agent capable of detecting DHDR protein ornucleic acid (e.g., mRNA, or genomic DNA) that encodes DHDR protein suchthat the presence of DHDR protein or nucleic acid is detected in thebiological sample. A preferred agent for detecting DHDR mRNA or genomicDNA is a labeled nucleic acid probe capable of hybridizing to DHDR mRNAor genomic DNA. The nucleic acid probe can be, for example, the DHDRnucleic acid set forth in SEQ ID NO:1, 3, 4, 6, 7, 9, 10, 12, 14, or 16,or the DNA insert of the plasmid deposited with ATCC as Accession Number______, PTA-1845, ______, ______, or ______, or a portion thereof, suchas an oligonucleotide of at least 15, 30, 50, 100, 250 or 500nucleotides in length and sufficient to specifically hybridize understringent conditions to DHDR mRNA or genomic DNA. Other suitable probesfor use in the diagnostic assays of the invention are described herein.

[0270] A preferred agent for detecting DHDR protein is an antibodycapable of binding to DHDR protein, preferably an antibody with adetectable label. Antibodies can be polyclonal, or more preferably,monoclonal. An intact antibody, or a fragment thereof (e.g., Fab orF(ab′)2) can be used. The term “labeled”, with regard to the probe orantibody, is intended to encompass direct labeling of the probe orantibody by coupling (i.e., physically linking) a detectable substanceto the probe or antibody, as well as indirect labeling of the probe orantibody by reactivity with another reagent that is directly labeled.Examples of indirect labeling include detection of a primary antibodyusing a fluorescently labeled secondary antibody and end-labeling of aDNA probe with biotin such that it can be detected with fluorescentlylabeled streptavidin. The term “biological sample” is intended toinclude tissues, cells and biological fluids isolated from a subject, aswell as tissues, cells and fluids present within a subject. That is, thedetection method of the invention can be used to detect DHDR mRNA,protein, or genomic DNA in a biological sample in vitro as well as invivo. For example, in vitro techniques for detection of DHDR mRNAinclude Northern hybridizations and in situ hybridizations. In vitrotechniques for detection of DHDR protein include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitation andimmunofluo rescence. In vitro techniques for detection of DHDR genomicDNA include Southern hybridizations. Furthermore, in vivo techniques fordetection of DHDR protein include introducing into a subject a labeledanti-DHDR antibody. For example, the antibody can be labeled with aradioactive marker whose presence and location in a subject can bedetected by standard imaging techniques.

[0271] In one embodiment, the biological sample contains proteinmolecules from the test subject. Alternatively, the biological samplecan contain mRNA molecules from the test subject or genomic DNAmolecules from the test subject. A preferred biological sample is aserum sample isolated by conventional means from a subject.

[0272] In another embodiment, the methods further involve obtaining acontrol biological sample from a control subject, contacting the controlsample with a compound or agent capable of detecting DHDR protein, mRNA,or genomic DNA, such that the presence of DHDR protein, mRNA or genomicDNA is detected in the biological sample, and comparing the presence ofDHDR protein, mRNA or genomic DNA in the control sample with thepresence of DHDR protein, mRNA or genomic DNA in the test sample.

[0273] The invention also encompasses kits for detecting the presence ofDHDR in a biological sample. For example, the kit can comprise a labeledcompound or agent capable of detecting DHDR protein or mRNA in abiological sample; means for determining the amount of DHDR in thesample; and means for comparing the amount of DHDR in the sample with astandard. The compound or agent can be packaged in a suitable container.The kit can further comprise instructions for using the kit to detectDHDR protein or nucleic acid.

[0274] 2. Prognostic Assays

[0275] The diagnostic methods described herein can furthermore beutilized to identify subjects having or at risk of developing a diseaseor disorder associated with aberrant or unwanted DHDR expression oractivity. As used herein, the term “aberrant” includes a DHDR expressionor activity which deviates from the wild type DHDR expression oractivity. Aberrant expression or activity includes increased ordecreased expression or activity, as well as expression or activitywhich does not follow the wild type developmental pattern of expressionor the subcellular pattern of expression. For example, aberrant DHDRexpression or activity is intended to include the cases in which amutation in the DHDR gene causes the DHDR gene to be under-expressed orover-expressed and situations in which such mutations result in anon-functional DHDR protein or a protein which does not function in awild-type fashion, e.g., a protein which does not interact with a DHDRsubstrate, or one which interacts with a non-DHDR substrate. As usedherein, the term “unwanted” includes an unwanted phenomenon involved ina biological response such as cellular proliferation. For example, theterm unwanted includes a DHDR expression or activity which isundesirable in a subject.

[0276] The assays described herein, such as the preceding diagnosticassays or the following assays, can be utilized to identify a subjecthaving or at risk of developing a disorder associated with amisregulation in DHDR protein activity or nucleic acid expression, suchas a CNS disorder (e.g., a cognitive or neurodegenerative disorder), acellular proliferation, growth, differentiation, or migration disorder,a cardiovascular disorder, musculoskeletal disorder, an immune disorder,a viral disorder, or a hormonal disorder. Alternatively, the prognosticassays can be utilized to identify a subject having or at risk fordeveloping a disorder associated with a misregulation in DHDR proteinactivity or nucleic acid expression, such as a CNS disorder, a cellularproliferation, growth, differentiation, or migration disorder, amusculoskeletal disorder, a cardiovascular disorder, an immune disorder,a viral disorder, or a hormonal disorder. Thus, the present inventionprovides a method for identifying a disease or disorder associated withaberrant or unwanted DHDR expression or activity in which a test sampleis obtained from a subject and DHDR protein or nucleic acid (e.g., mRNAor genomic DNA) is detected, wherein the presence of DHDR protein ornucleic acid is diagnostic for a subject having or at risk of developinga disease or disorder associated with aberrant or unwanted DHDRexpression or activity. As used herein, a “test sample” refers to abiological sample obtained from a subject of interest. For example, atest sample can be a biological fluid (e.g., cerebrospinal fluid orserum), cell sample, or tissue sample (e.g., a liver sample).

[0277] Furthermore, the prognostic assays described herein can be usedto determine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant or unwanted DHDR expression or activity. Forexample, such methods can be used to determine whether a subject can beeffectively treated with an agent for a CNS disorder, a musculardisorder, a cellular proliferation, growth, differentiation, ormigration disorder, an immune disorder, a viral disorder, or a hormonaldisorder. Thus, the present invention provides methods for determiningwhether a subject can be effectively treated with an agent for adisorder associated with aberrant or unwanted DHDR expression oractivity in which a test sample is obtained and DHDR protein or nucleicacid expression or activity is detected (e.g., wherein the abundance ofDHDR protein or nucleic acid expression or activity is diagnostic for asubject that can be administered the agent to treat a disorderassociated with aberrant or unwanted DHDR expression or activity).

[0278] The methods of the invention can also be used to detect geneticalterations in a DHDR gene, thereby determining if a subject with thealtered gene is at risk for a disorder characterized by misregulation inDHDR protein activity or nucleic acid expression, such as a CNSdisorder, a musculoskeletal disorder, a cellular proliferation, growth,differentiation, or migration disorder, a cardiovascular disorder, animmune disorder, a viral disorder, or a hormonal disorder. In preferredembodiments, the methods include detecting, in a sample of cells fromthe subject, the presence or absence of a genetic alterationcharacterized by at least one of an alteration affecting the integrityof a gene encoding a DHDR-protein, or the mis-expression of the DHDRgene. For example, such genetic alterations can be detected byascertaining the existence of at least one of 1) a deletion of one ormore nucleotides from a DHDR gene; 2) an addition of one or morenucleotides to a DHDR gene; 3) a substitution of one or more nucleotidesof a DHDR gene, 4) a chromosomal rearrangement of a DHDR gene; 5) analteration in the level of a messenger RNA transcript of a DHDR gene, 6)aberrant modification of a DHDR gene, such as of the methylation patternof the genomic DNA, 7) the presence of a non-wild type splicing patternof a messenger RNA transcript of a DHDR gene, 8) a non-wild type levelof a DHDR-protein, 9) allelic loss of a DHDR gene, and 10) inappropriatepost-translational modification of a DHDR-protein. As described herein,there are a large number of assays known in the art which can be usedfor detecting alterations in a DHDR gene. A preferred biological sampleis a tissue (e.g., a liver sample) or serum sample isolated byconventional means from a subject.

[0279] In certain embodiments, detection of the alteration involves theuse of a probe/primer in a polymerase chain reaction (PCR) (see, e.g.,U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR,or, alternatively, in a ligation chain reaction (LCR) (see, e.g.,Landegran et al. (1988) Science 241-1077-1080; and Nakazawa et al.(1994) Proc. Natl. Acad. Sci. USA 91:360-364), the latter of which canbe particularly useful for detecting point mutations in a DHDR gene (seeAbravaya et al. (1995) Nucleic Acids Res. 23:675-682). This method caninclude the steps of collecting a sample of cells from a subject,isolating nucleic acid (e.g., genomic, mRNA or both) from the cells ofthe sample, contacting the nucleic acid sample with one or more primerswhich specifically hybridize to a DHDR gene under conditions such thathybridization and amplification of the DHDR gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. It is anticipated that PCR and/or LCR may bedesirable to use as a preliminary amplification step in conjunction withany of the techniques used for detecting mutations described herein.

[0280] Alternative amplification methods include: self sustainedsequence replication (Guatelli, J. C. et al., (1990) Proc. Natl. Acad.Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D.Y. et al., (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-BetaReplicase (Lizardi, P. M. et al. (1988) Bio-Technology 6:1197), or anyother nucleic acid amplification method, followed by the detection ofthe amplified molecules using techniques well known to those of skill inthe art. These detection schemes are especially useful for the detectionof nucleic acid molecules if such molecules are present in very lownumbers.

[0281] In an alternative embodiment, mutations in a DHDR gene from asample cell can be identified by alterations in restriction enzymecleavage patterns. For example, sample and control DNA is isolated,amplified (optionally), digested with one or more restrictionendonucleases, and fragment length sizes are determined by gelelectrophoresis and compared. Differences in fragment length sizesbetween sample and control DNA indicates mutations in the sample DNA.Moreover, the use of sequence specific ribozymes (see, for example, U.S.Pat. No. 5,498,531) can be used to score for the presence of specificmutations by development or loss of a ribozyme cleavage site.

[0282] In other embodiments, genetic mutations in DHDR can be identifiedby hybridizing a sample and control nucleic acids, e.g., DNA or RNA, tohigh density arrays containing hundreds or thousands of oligonucleotidesprobes (Cronin, M. T. et al. (1996) Hum. Mutat. 7:244-255; Kozal, M. J.et al. (1996) Nat. Med. 2:753-759). For example, genetic mutations inDHDR can be identified in two dimensional arrays containinglight-generated DNA probes as described in Cronin et al. (1996) supra.Briefly, a first hybridization array of probes can be used to scanthrough long stretches of DNA in a sample and control to identify basechanges between the sequences by making linear arrays of sequentialoverlapping probes. This step allows the identification of pointmutations. This step is followed by a second hybridization array thatallows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

[0283] In yet another embodiment, any of a variety of sequencingreactions known in the art can be used to directly sequence the DHDRgene and detect mutations by comparing the sequence of the sample DHDRwith the corresponding wild-type (control) sequence. Examples ofsequencing reactions include those based on techniques developed byMaxam and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplatedthat any of a variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays ((1995) Biotechniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT InternationalPublication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr.36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol.38:147-159).

[0284] Other methods for detecting mutations in the DHDR gene includemethods in which protection from cleavage agents is used to detectmismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al.(1985) Science 230:1242). In general, the art technique of “mismatchcleavage” starts by providing heteroduplexes of formed by hybridizing(labeled) RNA or DNA containing the wild-type DHDR sequence withpotentially mutant RNA or DNA obtained from a tissue sample. Thedouble-stranded duplexes are treated with an agent which cleavessingle-stranded regions of the duplex such as which will exist due tobasepair mismatches between the control and sample strands. Forinstance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybridstreated with S1 nuclease to enzymatically digesting the mismatchedregions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can betreated with hydroxylamine or osmium tetroxide and with piperidine inorder to digest mismatched regions. After digestion of the mismatchedregions, the resulting material is then separated by size on denaturingpolyacrylamide gels to determine the site of mutation. See, for example,Cotton et al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al.(1992) Methods Enzymol. 217:286-295. In a preferred embodiment, thecontrol DNA or RNA can be labeled for detection.

[0285] In still another embodiment, the mismatch cleavage reactionemploys one or more proteins that recognize mismatched base pairs indouble-stranded DNA (so called “DNA mismatch repair” enzymes) in definedsystems for detecting and mapping point mutations in DHDR cDNAs obtainedfrom samples of cells. For example, the mutY enzyme of E. coli cleaves Aat G/A mismatches and the thymidine DNA glycosylase from HeLa cellscleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis15:1657-1662). According to an exemplary embodiment, a probe based on aDHDR sequence, e.g., a wild-type DHDR sequence, is hybridized to a cDNAor other DNA product from a test cell(s). The duplex is treated with aDNA mismatch repair enzyme, and the cleavage products, if any, can bedetected from electrophoresis protocols or the like. See, for example,U.S. Pat. No. 5,459,039.

[0286] In other embodiments, alterations in electrophoretic mobilitywill be used to identify mutations in DHDR genes. For example, singlestrand conformation polymorphism (SSCP) may be used to detectdifferences in electrophoretic mobility between mutant and wild typenucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA; 86:2766,see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992)Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments ofsample and control DHDR nucleic acids will be denatured and allowed torenature. The secondary structure of single-stranded nucleic acidsvaries according to sequence, the resulting alteration inelectrophoretic mobility enables the detection of even a single basechange. The DNA fragments may be labeled or detected with labeledprobes. The sensitivity of the assay may be enhanced by using RNA(rather than DNA), in which the secondary structure is more sensitive toa change in sequence. In a preferred embodiment, the subject methodutilizes heteroduplex analysis to separate double stranded heteroduplexmolecules on the basis of changes in electrophoretic mobility (Keen etal. (1991) Trends Genet. 7:5).

[0287] In yet another embodiment the movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (DGGE) (Myers etal. (1985) Nature 313:495). When DGGE is used as the method of analysis,DNA will be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys. Chem. 265:12753).

[0288] Examples of other techniques for detecting point mutationsinclude, but are not limited to, selective oligonucleotidehybridization, selective amplification, or selective primer extension.For example, oligonucleotide primers may be prepared in which the knownmutation is placed centrally and then hybridized to target DNA underconditions which permit hybridization only if a perfect match is found(Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl.Acad. Sci. USA 86:6230). Such allele specific oligonucleotides arehybridized to PCR amplified target DNA or a number of differentmutations when the oligonucleotides are attached to the hybridizingmembrane and hybridized with labeled target DNA.

[0289] Alternatively, allele specific amplification technology whichdepends on selective PCR amplification may be used in conjunction withthe instant invention. Oligonucleotides used as primers for specificamplification may carry the mutation of interest in the center of themolecule (so that amplification depends on differential hybridization)(Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme3′ end of one primer where, under appropriate conditions, mismatch canprevent, or reduce polymerase extension (Prossner (1993) Tibtech11:238). In addition it may be desirable to introduce a novelrestriction site in the region of the mutation to create cleavage-baseddetection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It isanticipated that in certain embodiments amplification may also beperformed using Taq ligase for amplification (Barany (1991) Proc. Natl.Acad. Sci USA 88:189). In such cases, ligation will occur only if thereis a perfect match at the 3′ end of the 5′ sequence making it possibleto detect the presence of a known mutation at a specific site by lookingfor the presence or absence of amplification.

[0290] The methods described herein may be performed, for example, byutilizing pre-packaged diagnostic kits comprising at least one probenucleic acid or antibody reagent described herein, which may beconveniently used, e.g., in clinical settings to diagnose patientsexhibiting symptoms or family history of a disease or illness involvinga DHDR gene.

[0291] Furthermore, any cell type or tissue in which DHDR is expressedmay be utilized in the prognostic assays described herein.

[0292] 3. Monitoring of Effects During Clinical Trials

[0293] Monitoring the influence of agents (e.g., drugs) on theexpression or activity of a DHDR protein (e.g., the modulation of avirus activity and/or the modulation of cell proliferation and/ormigration) can be applied not only in basic drug screening, but also inclinical trials. For example, the effectiveness of an agent determinedby a screening assay as described herein to increase DHDR geneexpression, protein levels, or upregulate DHDR activity, can bemonitored in clinical trials of subjects exhibiting decreased DHDR geneexpression, protein levels, or downregulated DHDR activity.Alternatively, the effectiveness of an agent determined by a screeningassay to decrease DHDR gene expression, protein levels, or downregulateDHDR activity, can be monitored in clinical trials of subjectsexhibiting increased DHDR gene expression, protein levels, orupregulated DHDR activity. In such clinical trials, the expression oractivity of a DHDR gene, and preferably, other genes that have beenimplicated in, for example, a DHDR-associated disorder can be used as a“read out” or markers of the phenotype of a particular cell.

[0294] For example, and not by way of limitation, genes, including DHDR,that are modulated in cells by treatment with an agent (e.g., compound,drug or small molecule) which modulates DHDR activity (e.g., identifiedin a screening assay as described herein) can be identified. Thus, tostudy the effect of agents on DHDR-associated disorders (e.g., disorderscharacterized by viral infection and/or deregulated cell proliferationand/or migration), for example, in a clinical trial, cells can beisolated and RNA prepared and analyzed for the levels of expression-ofDHDR and other genes implicated in the DHDR-associated disorder,respectively. The levels of gene expression (e.g., a gene expressionpattern) can be quantified by northern-blot analysis or RT-PCR, asdescribed herein, or alternatively by measuring the amount of proteinproduced, by one of the methods as described herein, or by measuring thelevels of activity of DHDR or other genes. In this way, the geneexpression pattern can serve as a marker, indicative of thephysiological response of the cells to the agent. Accordingly, thisresponse state may be determined before, and at various points duringtreatment of the individual with the agent.

[0295] In a preferred embodiment, the present invention provides amethod for monitoring the effectiveness of treatment of a subject withan agent (e.g., an agonist, antagonist, peptidomimetic, protein,peptide, nucleic acid, small molecule, or other drug candidateidentified by the screening assays described herein) including the stepsof (i) obtaining a pre-administration sample from a subject prior toadministration of the agent; (ii) detecting the level of expression of aDHDR protein, mRNA, or genomic DNA in the preadministration sample;(iii) obtaining one or more post-administration samples from thesubject; (iv) detecting the level of expression or activity of the DHDRprotein, mRNA, or genomic DNA in the post-administration samples; (v)comparing the level of expression or activity of the DHDR protein, mRNA,or genomic DNA in the pre-administration sample with the DHDR protein,mRNA, or genomic DNA in the post administration sample or samples; and(vi) altering the administration of the agent to the subjectaccordingly. For example, increased administration of the agent may bedesirable to increase the expression or activity of DHDR to higherlevels than detected, i.e., to increase the effectiveness of the agent.Alternatively, decreased administration of the agent may be desirable todecrease expression or activity of DHDR to lower levels than detected,i.e., to decrease the effectiveness of the agent. According to such anembodiment, DHDR expression or activity may be used as an indicator ofthe effectiveness of an agent, even in the absence of an observablephenotypic response.

[0296] D. Methods of Treatment:

[0297] The present invention provides for both prophylactic andtherapeutic methods of treating a subject at risk of (or susceptible to)a disorder or having a disorder associated with aberrant or unwantedDHDR expression or activity, e.g., a dehydrogenase-associated disordersuch as a CNS disorder; a cellular proliferation, growth,differentiation, or migration disorder; a, musculoskeletal disorder; acardiovascular disorder; an immune disorder; a viral disorder; or ahormonal disorder. As used herein, “treatment” of a subject includes theapplication or administration of a therapeutic agent to a subject, orapplication or administration of a therapeutic agent to a cell or tissuefrom a subject, who has a diseases or disorder, has a symptom of adisease or disorder, or is at risk of (or susceptible to) a disease ordisorder, with the purpose to cure, heal, alleviate, relieve, alter,remedy, ameliorate, improve, or affect the disease or disorder, thesymptom of the disease or disorder, or the risk of (or susceptibilityto) the disease or disorder. As used herein, a “therapeutic agent”includes, but is not limited to, small molecules, peptides,polypeptides, antibodies, ribozymes, and antisense oligonucleotides.

[0298] With regard to both prophylactic and therapeutic methods oftreatment, such treatments may be specifically tailored or modified,based on knowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (erg., a patient's “drugresponse phenotype”, or “drug response genotype”). Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the DHDR molecules ofthe present invention or DHDR modulators according to that individual'sdrug response genotype. Pharmacogenomics allows a clinician or physicianto target prophylactic or therapeutic treatments to patients who willmost benefit from the treatment and to avoid treatment of patients whowill experience toxic drug-related side effects.

[0299] 1. Prophylactic Methods

[0300] In one aspect, the invention provides a method for preventing ina subject, a disease or condition associated with an aberrant orunwanted DHDR expression or activity, by administering to the subject aDHDR or an agent which modulates DHDR expression or at least one DHDRactivity. Subjects at risk for a disease which is caused or contributedto by aberrant or unwanted DHDR expression or activity can be identifiedby, for example, any or a combination of diagnostic or prognostic assaysas described herein. Administration of a prophylactic agent can occurprior to the manifestation of symptoms characteristic of the DHDRaberrancy, such that a disease or disorder is prevented or,alternatively, delayed in its progression. Depending on the type of DHDRaberrancy, for example, a DHDR, DHDR agonist or DHDR antagonist agentcan be used for treating the subject. The appropriate agent can bedetermined based on screening assays described herein.

[0301] 2. Therapeutic Methods

[0302] Another aspect of the invention pertains to methods of modulatingDHDR expression or activity for therapeutic purposes. Accordingly, in anexemplary embodiment, the modulatory method of the invention involvescontacting a cell with a DHDR or agent that modulates one or more of theactivities of DHDR protein activity associated with the cell. An agentthat modulates DHDR protein-activity can be an agent as describedherein, such as a nucleic acid or a protein, a naturally-occurringtarget molecule of a DHDR protein (e-g., a DHDR substrate), a DHDRantibody, a DHDR agonist or antagonist, a peptidomimetic of a DHDRagonist or antagonist, or other small molecule. In one embodiment, theagent stimulates one or more DHDR activities. Examples of suchstimulatory agents include active DHDR protein and a nucleic acidmolecule encoding DHDR that has been introduced into the cell. Inanother embodiment, the agent inhibits one or more DHDR activities.Examples of such inhibitory agents include antisense DHDR nucleic acidmolecules, anti-DHDR antibodies, and DHDR inhibitors. These modulatorymethods can be performed in vitro (e.g., by culturing the cell with theagent) or, alternatively, in vivo (e.g., by administering the agent to asubject). As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant or unwanted expression or activity of a DHDR protein or nucleicacid molecule. In one embodiment, the method involves administering anagent (e.g., an agent identified by a screening assay described herein),or combination of agents that modulates (e.g., upregulates ordownregulates) DHDR expression or activity. In another embodiment, themethod involves administering a DHDR protein or nucleic acid molecule astherapy to compensate for reduced, aberrant, or unwanted DHDR expressionor activity

[0303] Stimulation of DHDR activity is desirable in situations in whichDHDR is abnormally downregulated and/or in which increased DHDR activityis likely to have a beneficial effect. Likewise, inhibition of DHDRactivity is desirable in situations in which DHDR is abnormallyupregulated and/or in which decreased DHDR activity is likely to have abeneficial effect.

[0304] 3. Pharmacogenomics

[0305] The DHDR molecules of the present invention, as well as agents,or modulators which have a stimulatory or inhibitory effect on DHDRactivity (e.g., DHDR gene expression) as identified by a screening assaydescribed herein can be administered to individuals to treat(prophylactically or therapeutically) DHDR-associated disorders (e.g.,proliferative disorders, CNS disorders, cardiac disorders, metabolicdisorders, or muscular disorders) associated with aberrant or unwantedDHDR activity. In conjunction with such treatment, pharmacogenomics(i.e., the study of the relationship between an individual's genotypeand that individual's response to a foreign compound or drug) may beconsidered. Differences in metabolism of therapeutics can lead to severetoxicity or therapeutic failure by altering the relation between doseand blood concentration of the pharmacologically active drug. Thus, aphysician or clinician may consider applying knowledge obtained inrelevant pharmacogenomics studies in determining whether to administer aDHDR molecule or DHDR modulator as well as tailoring the dosage and/ortherapeutic regimen of treatment with a DHDR molecule or DHDR modulator.

[0306] Pharmacogenomics deals with clinically significant hereditaryvariations in the response to drugs due to altered drug disposition andabnormal action in affected persons. See, for example, Eichelbaum, M. etal. (1996) Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 and Linder,M. W. el al. (1997) Clin. Chem. 43(2):254-266. In general, two types ofpharmacogenetic conditions can be differentiated. Genetic conditionstransmitted as a single factor altering the way drugs act on the body(altered drug action) or genetic conditions transmitted as singlefactors altering the way the body acts on drugs (altered drugmetabolism). These pharmacogenetic conditions can occur either as raregenetic defects or as naturally-occurring polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

[0307] One pharmacogenomics approach to identifying genes that predictdrug response, known as “a genome-wide association”, relies primarily ona high-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants). Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

[0308] Alternatively, a method termed the “candidate gene approach”, canbe utilized to identify genes that predict drug response. According tothis method, if a gene that encodes a drugs target is known (e.g., aDHDR protein of the present invention), all common variants of that genecan be fairly easily identified in the population and it can bedetermined if having one version of the gene versus another isassociated with a particular drug response.

[0309] As an illustrative embodiment, the activity of drug metabolizingenzymes is a major determinant of both the intensity and duration ofdrug action. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

[0310] Alternatively, a method termed the “gene expression profiling”can be utilized to identify genes that predict drug response. Forexample, the gene expression of an animal dosed with a drug (e.g., aDHDR molecule or DHDR modulator of the present invention) can give anindication whether gene pathways related to toxicity have been turnedon.

[0311] Information generated from more than one of the abovepharmacogenomics approaches can be used to determine appropriate dosageand treatment regimens for prophylactic or therapeutic treatment anindividual. This knowledge, when applied to dosing or drug selection,can avoid adverse reactions or therapeutic failure and thus enhancetherapeutic or prophylactic efficiency when treating a subject with aDHDR molecule or DHDR modulator, such as a modulator identified by oneof the exemplary screening assays described herein.

[0312] E. Electronic Apparatus Readable Media and Arrays

[0313] Electronic apparatus readable media-comprising DHDR sequenceinformation is also provided. As used herein, “DHDR sequenceinformation” refers to any nucleotide and/or amino acid sequenceinformation particular to the DHDR molecules of the present invention,including but not limited to full-length nucleotide and/or amino acidsequences, partial nucleotide and/or amino acid sequences, polymorphicsequences including single nucleotide polymorphisms (SNPs), epitopesequences, and the like. Moreover, information “related to” said DHDRsequence information includes detection of the presence or absence of asequence (e.g., detection of expression of a sequence, fragment,polymorphism, etc.), determination of the level of a sequence (e.g.,detection of a level of expression, for example, a quantitativedetection), detection of a reactivity to a sequence (e.g., detection ofprotein expression and/or levels, for example, using a sequence-specificantibody), and the like. As used herein, “electronic apparatus readablemedia” refers to any suitable medium for storing, holding, or containingdata or information that can be read and accessed directly by anelectronic apparatus. Such media can include, but are not limited to:magnetic storage media, such as floppy discs, hard disc storage medium,and magnetic tape; optical storage media such as compact discs;electronic storage media such as RAM, ROM, EPROM, EEPROM and the like;and general hard disks and hybrids of these categories such asmagnetic/optical storage media. The medium is adapted or configured forhaving recorded thereon DHDR sequence information of the presentinvention.

[0314] As used herein, the term “electronic apparatus” is intended toinclude any suitable computing or processing apparatus or other deviceconfigured or adapted for storing data or information. Examples ofelectronic apparatus suitable for use with the present invention includestand-alone computing apparatuses; networks, including a local areanetwork (LAN), a wide area network (WAN) Internet, Intranet, andExtranet; electronic appliances such as a personal digital assistants(PDAs), cellular phone, pager and the like; and local and distributedprocessing systems.

[0315] As used herein, “recorded” refers to a process for storing orencoding information on the electronic apparatus readable medium. Thoseskilled in the art can readily adopt any of the presently known methodsfor recording information on known media to generate manufacturescomprising the DHDR sequence information.

[0316] A variety of software programs and formats can be used to storethe sequence information on the electronic apparatus readable medium.For example, the sequence information can be represented in a wordprocessing text file, formatted in commercially-available software suchas WordPerfect and Microsoft Word, represented in the form of an ASCIIfile, or stored in a database application, such as DB2, Sybase, Oracle,or the like, as well as in other forms. Any number of dataprocessorstructuring formats (e.g., text file or database) may be employed inorder to obtain or create a medium having recorded thereon the DHDRsequence information.

[0317] By providing DHDR sequence information in readable form, one canroutinely access the sequence information for a variety of purposes. Forexample, one skilled in the art can use the sequence information inreadable form to compare a target sequence or target structural motifwith the sequence information stored within the data storage means.Search means are used to identify fragments or regions of the sequencesof the invention which match a particular target sequence or targetmotif.

[0318] The present invention therefore provides a medium for holdinginstructions for performing a method for determining whether a subjecthas a DHDR associated disease or disorder or a pre-disposition to a DHDRassociated disease or disorder, wherein the method comprises the stepsof determining DHDR sequence information associated with the subject andbased on the DHDR sequence information, determining whether-the subjecthas a DHDR associated disease or disorder or a pre-disposition to a DHDRassociated disease or disorder, and/or recommending a particulartreatment for the disease, disorder, or predisease condition.

[0319] The present invention further provides in an electronic systemand/or in a network, a method for determining whether a subject has aDHDR associated disease or disorder or a pre-disposition to a diseaseassociated with DHDR wherein the method comprises the steps ofdetermining DHDR sequence information associated with the subject, andbased on the DHDR sequence information, determining whether the subjecthas a DHDR associated disease or disorder or a pre-disposition to a DHDRassociated disease or disorder, and/or recommending a particulartreatment for the disease, disorder or pre-disease condition. The methodmay further comprise the step of receiving phenotypic informationassociated with the subject and/or acquiring from a network phenotypicinformation associated with the subject.

[0320] The present invention also provides in a network, a method fordetermining whether a subject has a DHDR associated disease or disorderor a pre-disposition to a DHDR associated disease or disorder associatedwith DHDR, said method comprising the steps of receiving DHDR sequenceinformation from the subject and/or information related thereto,receiving phenotypic information associated with the subject, acquiringinformation from the network corresponding to DHDR and/or a DHDRassociated disease or disorder, and based on one or more of thephenotypic information, the DHDR information (e.g., sequence informationand/or information related thereto), and the acquired information,determining whether the subject has a DHDR associated disease ordisorder or a pre-disposition to a DHDR associated disease or disorder.The method may further comprise the step of recommending a particulartreatment for the disease, disorder or pre-disease condition.

[0321] The present invention also provides a business method fordetermining whether a subject has a DHDR associated disease or disorderor a pre-disposition to a DHDR associated disease or disorder, saidmethod comprising the steps of receiving information related to DHDR(e.g., sequence information and/or information related thereto),receiving phenotypic information associated with the subject, acquiringinformation from the network related to DHDR and/or related to a DHDRassociated disease or disorder, and based on one or more of thephenotypic information, the DHDR information, and the acquiredinformation, determining whether the subject has a DHDR associateddisease or disorder or a pre-disposition to a DHDR associated disease ordisorder. The method may further comprise the step of recommending aparticular treatment for the disease, disorder or pre-disease condition.

[0322] The invention also includes an array comprising a DHDR sequenceof the present invention. The array can be used to assay expression ofone or more genes in the array. In one embodiment the array can be usedto assay gene expression in a tissue to ascertain tissue specificity ofgenes in the array. In this manner, up to about 7600 genes can besimultaneously assayed for expression, one of which can be DHDR. Thisallows a profile to be developed showing a battery of genes specificallyexpressed in one or more tissues.

[0323] In addition to such qualitative determination, the inventionallows the quantitation of gene expression. Thus, not only tissuespecificity, but also the level of expression of a battery of genes inthe tissue is ascertainable. Thus, genes can be grouped on the basis oftheir tissue expression per se and level of expression in that tissue.This is useful, for example, in ascertaining the relationship of geneexpression between or among tissues. Thus, one tissue can be perturbedand the effect on gene expression in a second tissue can be determined.In this context, the effect of one cell type on another cell type inresponse to a biological stimulus can be determined. Such adetermination is useful, for example, to know the effect of cell-cellinteraction at the level of gene expression. If an agent is administeredtherapeutically to treat one cell type but has an undesirable effect onanother cell type, the invention provides an assay to determine themolecular basis of the undesirable effect and thus provides theopportunity to co-administer a counteracting agent or otherwise treatthe undesired effect. Similarly, even within a single cell type,undesirable biological effects can be determined at the molecular level.Thus, the effects of an agent on expression of other than the targetgene can be ascertained and counteracted.

[0324] In another embodiment, the array can be used to monitor the timecourse of expression of one or more genes in the array. This can occurin various biological contexts, as disclosed herein, for exampledevelopment of a DHDR associated disease or disorder, progression ofDHDR associated disease or disorder, and processes, such a cellulartransformation associated with the DHDR associated disease or disorder.

[0325] The array is also useful for ascertaining the effect of theexpression of a gene on the expression of other genes in the same cellor in different cells (e.g., ascertaining the effect of DHDR expressionon the expression of other genes). This provides, for example, for aselection of alternate molecular targets for therapeutic intervention ifthe ultimate or downstream target cannot be regulated.

[0326] The array is also useful for ascertaining differential expressionpatterns of one or more genes in normal and abnormal cells. Thisprovides a battery of genes (e.g., including DHDR) that could serve as amolecular target for diagnosis or therapeutic intervention.

[0327] This invention is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patents, and published patent applications cited throughoutthis application, as well as the figures and the sequence listing, areincorporated herein by reference.

EXAMPLES Example 1

[0328] Identification and Characterization of Human and Mouse DHDR cDNA

[0329] In this example, the identification and characterization of thegenes encoding human DHDR-1 (clone Fbh32142), DHDR-2 (clone Fbh21481),DHDR-3 (clone Fbh25964), and DHDR-4 (clone Fbh21686), and mouse DHDR-2(clone m21481) is described.

[0330] Isolation of the DHDR cDNA

[0331] The invention is based, at least in part, on the discovery ofseveral human and mouse genes encoding novel proteins, referred toherein as DHDR-1, DHDR-2, DHDR-3 and DHDR-4. The entire sequences ofhuman clones Fbh32142, Fbh21481, Fbh25964, and Fbh21686, and mouse clonem21481 were determined and found to contain open reading frames termedhuman “DHDR-1”, “DHDR-2”, “DHDR-3”, and “DHDR-4”, and mouse “DHDR-2”,respectively, set forth in FIGS. 1, 6, 11, 16, and 31A, respectively.The amino acid sequences of these human and mouse DHDR expressionproducts are set forth in FIGS. 1, 6, 11, 16, and 31B, respectively. Thehuman DHDR-1 protein sequence set forth in SEQ ID NO:2 comprises about802 amino acid residues and is shown in FIGS. 1A-1D. The human DHDR-2protein sequence set forth in SEQ ID NO:5 comprises about 311 amino acidresidues and is shown in FIGS. 6A-6B. The human DHDR-3 protein sequenceset forth in SEQ ID NO:8 comprises about 369 amino acid residues and isshown in FIGS. 11A-11B. The human DHDR-4 protein sequence set forth inSEQ ID NO:11 comprises about 322 amino acid residues and is shown inFIG. 16. The mouse DHDR-2 protein set forth in SEQ ID NO:15 comprisesabout 311 amino acid residues and is shown in FIG. 31B. The codingregions (open reading frames) of SEQ ID NOs:1, 4, 7, 10, and 14 are setforth as SEQ ID NOs:3, 6, 9, 12, and 16. Clones Fbh32142, Fbh21481,Fbh25964, Fbh21686, and m21481, comprising the coding regions of humanDHDR-1, DHDR-2, DHDR-3, and DHDR-4, and mouse DHDR-2, respectively, weredeposited with the American Type Culture Collection (ATCC®), 10801University Boulevard, Manassas, Va. 20110-2209, on ______, May 9, 2000,______, Mar. 22, 2001, and ______, respectively, and assigned AccessionNos. ______, PTA-1845, ______, ______, and ______, respectively.

[0332] Analysis of the Human and Mouse DHDR Molecules

[0333] The amino acid sequences of human DHDR-1, DHDR-2, DHDR-3, andDHDR-4 were analyzed using the program PSORT (available online throughthe PSORT server website) to predict the localization of the proteinswithin the cell. This program assesses the presence of differenttargeting and localization amino acid sequences within the querysequence. The results of the analyses show that human DHDR-1 (SEQ IDNO:2) may be localized to the mitochondrion, to the endoplasmicreticulum, to the nucleus, or to secretory vesicles. The results of theanalyses further show that human DHDR-2 (SEQ ID NO:5) may be localizedto the mitochondrion, to the cytoplasm, to extracellular spaces or thecell wall, to vacuoles, to the nucleus, or to the endoplasmic reticulum.The results of the analyses further show that human DHDR-3 (SEQ ID NO:8)may be localized to the cytoplasm, to the mitochondrion, to the Golgi,to the endoplasmic reticulum, to the extracellular space or cell wall,to vacuoles, to the nucleus, or to secretory vesicles. The results ofthe analyses further show that human DHDR-4 (SEQ ID NO: 11) may belocalized to the nucleus, the cytoplasm, to the Golgi, to themitochondrion, to peroxisomes, to the endoplasmic reticulum, or tosecretory vesicles.

[0334] An alignment of the human DHDR-4 amino acid sequence with theamino acid sequence of Rattus norvegicus putative short-chaindehydrogenase/reductase (Accession Number AF099742) using the CLUSTAL W(1.74) multiple sequence alignment program is set forth in FIG. 17.

[0335] Each of the amino acid sequences of human DHDR-1, DHDR-2, DHDR-3,and DHDR-4 were analyzed by the SignalP program (Henrik et al. (1997)Prot. Eng. 10:1-6) for the presence of a signal peptide. These analysesrevealed the presence of a signal peptide in the amino acid sequence ofhuman DHDR-2 from residues 1-18 (FIG. 8). These analyses furtherrevealed the possible presence of a signal peptide in the amino acidsequence of human DHDR-4, from residues 1-19 (FIG. 19).

[0336] Searches of each of the amino acid sequences of human DHDR-1,DHDR-2, DHDR-3, and DHDR-4 were performed against the Memsat database(FIGS. 3, 8, 13, and 19). These searches resulted in the identificationof one transmembrane domain in the amino acid sequence of human DHDR-1(SEQ ID NO:2) at about residues 159-175, and one transmembrane domain inthe amino acid sequence of human DHDR-2 (SEQ ID NO:5) at about residues7-23 in the native molecule, or about residues 265-283 of the predictedmature protein. These searches further identified four transmembranedomains in the amino acid sequence of human DHDR-3 (SEQ ID NO:8) atabout residues 10-26, 73-90, 289-305, and 312-333, and fourtransmembrane domains in the amino acid sequence of human DHDR-4 (SEQ IDNO:11) at about residues 29-50, 170-188, 208-224, and 258-275 of thenative molecule, and at about residues 10-31, 151-169, 189-205, and239-256 of the predicted mature protein.

[0337] Searches of each of the amino acid sequences of human DHDR-1,DHDR-2, DHDR-3, and DHDR-4 were also performed against the HMM database(FIGS. 4, 9, 14, and 20). These searches resulted in the identificationof an “aldehyde dehydrogenase family domain” in the amino acid sequenceof human DHDR-1 (SEQ ID NO:2) at about residues 47-494 (score=149.8)(FIG. 4); the identification of a “short-chain dehydrogenase domain” inthe amino acid sequence of human DHDR-2 (SEQ ID NO:5) at about residues38-227 (score=120.0) (FIG. 9), and the identification of a “3-betahydroxysteroid dehydrogenase domain” at about residues 1-365(score=676.9), a “short chain dehydrogenase domain” at about residues10-197, and a “NAD-dependent epimerase/dehydratase domain” at aboutresidues 12-365 of the amino acid sequence of human DHDR-3 (SEQ ID NO:8)(FIGS. 14A-14B). These searches further resulted in the identificationof a “short chain dehydrogenase domain” at about residues 38-226(score=162.5), and a “short chain dehydrogenase/reductase domain” atabout residues 250-280 (score=47.2) of the amino acid sequence of humanDHDR-4 (SEQ ID NO:11) (FIG. 20).

[0338] Searches of each of the amino acid sequences of human DHDR-1,DHDR-2, DHDR-3, and DHDR-4 were also performed against the ProDomdatabase (FIGS. 5, 10, 15, and 21). These searches resulted in theidentification of an “aldehyde dehydrogenase oxidoreductase domain” inthe amino acid sequence of human DHDR-1 (SEQ ID NO:2) at about residues101-770 (score=280) (FIGS. 5A-5B), and the identification of an“oxidoreductase protein dehydrogenase domain” in the amino acid sequenceof human DHDR-2 (SEQ ID NO:5) at about residues 99-219 (score=113) (FIG.10). These searches further resulted in the identification of a “3-betahydroxysteroid dehydrogenase domain” in the amino acid sequence of humanDHDR-3 (SEQ ID NO:8) at about residues 11-362 (score=395) (FIG. 15).These searches further resulted in the identification of an“oxidoreductase protein dehydrogenase domain” at about residues 37-231(score=157), a “shikimate 5-dehydrogenase domain” at about residues35-82 (score=86), a “dehydrogenase domain” at about residues 237-286(score=84), and a “glucose-1-dehydrogenase domain” at about residues243-287 (score=92) of the amino acid sequence of human DHDR-4 (SEQ IDNO:11) (FIGS. 21A-21B).

[0339] The nucleotide sequences of the mouse and human DHDR-2 genes (SEQID NOs:14 and 4, respectively) were aligned using the GAP program in theGCG software package (nwsgapdna.cmp matrix) with a gap weight of 12 anda length weight of 4. The results are shown in FIGS. 32A-32B. As shownin the alignment, the mouse and human DHDR-2 nucleotide sequences areabout 88.1% identical.

[0340] The amino acid sequences of the mouse and human DHDR-2 genes (SEQID NOs:15 and 5, respectively) were aligned FIG. 33 depicts an alignmentof the mouse DHDR-2 amino acid sequence using the GAP program in the GCGsoftware package (Blosum 62 matrix) with a gap weight of 12 and a lengthweight of 4. The results are shown in FIG. 33. As shown in thealignment, the mouse and human DHDR-2 amino acid sequences are about91.3% identical.

[0341] Analysis of DHDR mRNA Expression

[0342] This example describes the expression of human DHDR mRNA invarious tissues, cell lines, and disease models, as determined using theTaqMan™ procedure and in situ hybridization analysis.

[0343] The Taqman™ procedure is a quantitative, real-time PCR-basedapproach to detecting mRNA. The RT-PCR reaction exploits the 5′ nucleaseactivity of AmpliTaq Gold™ DNA Polymerase to cleave a TaqMan™ probeduring PCR. Briefly, cDNA was generated from the samples of interest andserved as the starting material for PCR amplification. In addition tothe 5′ and 3′ gene-specific primers, a gene-specific oligonucleotideprobe (complementary to the region being amplified) was included in thereaction (i.e., the Taqman™ probe). The TaqMan™ probe included anoligonucleotide with a fluorescent reporter dye covalently linked to the5′ end of the probe (such as FAM (6-carboxyfluorescein), TET(6-carboxy-4,7,2′,7′-tetrachlorofluorescein), JOE(6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein), or VIC) and aquencher dye (TAMRA (6-carboxy-N,N,N′,N′-tetramethylrhodamine) at the 3′end of the probe.

[0344] During the PCR reaction, cleavage of the probe separated thereporter dye and the quencher dye, resulting in increased fluorescenceof the reporter. Accumulation of PCR products was detected directly bymonitoring the increase in fluorescence of the reporter dye. When theprobe was intact, the proximity of the reporter dye to the quencher dyeresulted in suppression of the reporter fluorescence. During PCR, if thetarget of interest was present, the probe specifically annealed betweenthe forward and reverse primer sites. The 5′-3′ nucleolytic activity ofthe AmpliTaq™ Gold DNA Polymerase cleaved the probe between the reporterand the quencher only if the probe hybridized to the target. The probefragments were then displaced from the target, and polymerization of thestrand continued. The 3′ end of the probe was blocked to preventextension of the probe during PCR. This process occurred in every cycleand did not interfere with the exponential accumulation of product. RNAwas prepared using the trizol method and treated with DNase to removecontaminating genomic DNA. cDNA was synthesized using standardtechniques. Mock cDNA synthesis in the absence of reverse transcriptaseresulted in samples with no detectable PCR amplification of the controlGAPDH or β-actin gene confirming efficient removal of genomic DNAcontamination.

[0345] For in situ analysis, various tissues, e.g., tissues obtainedfrom liver or colon, were first frozen on dry ice. Ten-micrometer-thicksections of the tissues were postfixed with 4% formaldehyde in DEPCtreated 1×phosphate-buffered saline (PBS) at room temperature for 10minutes before being rinsed twice in DEPC 1×phosphate-buffered salineand once in 0.1 M triethanolamine-HCl (pH 8.0). Following incubation in0.25% acetic anhydride-0.1 M triethanolamine-HCl for 10 minutes,sections were rinsed in DEPC 2×SSC (1×SSC is 0.15M NaCl plus 0.015Msodium citrate). Tissues were then dehydrated through a series ofethanol washes, incubated in 100% chloroform for 5 minutes, and thenrinsed in 100% ethanol for 1 minute and 95% ethanol for 1 minute andallowed to air dry.

[0346] Hybridizations were performed with ³⁵S-radiolabeled (5×10⁷cpm/ml) cRNA probes. Probes were incubated in the presence of a solutioncontaining 600 mM NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA, 0.01% shearedsalmon sperm DNA, 0.01% yeast tRNA, 0.05% yeast total RNA type X1,1×Denhardt's solution, 50% formamide, 10% dextran sulfate, 100 mMdithiothreitol, 0.1% sodium dodecyl sulfate (SDS), and 0.1% sodiumthiosulfate for 18 hours at 55° C.

[0347] After hybridization, slides were washed with 2×SSC. Sections werethen sequentially incubated at 37° C. in TNE (a solution containing 10mM Tris-HCl (pH 7.6), 500 mM NaCl, and 1 mM EDTA), for 10 minutes, inTNE with 10 μg of RNase A per ml for 30 minutes, and finally in TNE for10 minutes. Slides were then rinsed with 2×SSC at room temperature,washed with 2×SSC at 50° C. for 1 hour, washed with 0.2×SSC at 55° C.for 1 hour, and 0.2×SSC at 60° C. for 1 hour. Sections were thendehydrated rapidly through serial ethanol-0.3 M sodium acetateconcentrations before being air dried and exposed to Kodak Biomax MRscientific imaging film for 24 hours and subsequently dipped in NB-2photoemulsion and exposed at 4° C. for 7 days before being developed andcounter stained.

[0348] Human DHDR-1

[0349] The human DHDR-1 gene is highly expressed in coronary smoothmuscle cells (SMCs), human umbilical vein endothelial cells (HUVECs),kidney, skeletal muscle, adipose tissue, pancreas, skin, brain (cortexand hypothalamus), dorsal root ganglion (DRG), breast, ovary, prostate,prostate epithelial cells, colon, fibrotic liver, tonsil, and decubitusskin (see FIG. 22). The human DHDR-1 gene is expressed at lower levelsin a number of other tissues (see FIG. 22).

[0350] The human DHDR-1 gene is also expressed in a number oftumorigenic cell lines, including MCF-7 (breast tumor), ZR75 (breasttumor), T47D (breast tumor), MDA 231 (breast tumor), MDA 435 (breasttumor), SKBr3 (breast tumor), DLD1 (colon tumor—stage C), SW620 (colontumor—stage C), HCT116 (colon tumor), HT29 (colon tumor), Colo 205,NCIH125, NCIH322, NCIH460, A549, NHBE, SKOV-3 (ovary tumor), and OVCAR-3(ovary tumor).

[0351] Expression of the human DHDR-1 gene is upregulated in 3/5 ovarytumors (FIG. 23), as compared to normal ovary. In situ hybridizationexperiments also showed an upregulation of human DHDR-2 in 1 borderlineovary tumor and 2/2 invasive ovary tumors.

[0352] Expression of the human DHDR-1 gene is upregulated in 6/7 lungtumors (FIG. 23), as compared to normal lung. In situ hybridizationexperiments also showed an upregulation in 1/2 lung tumors.

[0353] Expression of the human DHDR-1 gene is upregulated in 6/15hyperplastic/dysplastic lesions in the colon (FIG. 24), 3/4 colon tumors(FIG. 23), and 12/13 colon tumor metastases to the liver (FIGS. 23 and24), as compared to the expression in normal colon and liver. In situhybridization experiments also showed a marked elevation of expressionas early as hyperplasia/dysplasia (1/1 hyperplastic/dysplastic lesions),which is maintained in primary tumors (5/5 tumors) and metastases (3/3metastases to the liver).

[0354] Expression of the human DHDR-1 gene is upregulated in NOCsynchronized HCT116 cells (colon tumor derived cell line) at the timepoint t=24 after entry into the cell cycle (FIG. 25). The time point t=0signifies the G2/M border.

[0355] Human DHDR-2

[0356] Expression of the human DHDR-2 gene is downregulated in 7/7breast tumors (FIG. 26), as compared to the expression in normal breast;downregulated in 6/6 colon tumors and 4/4 colon tumor metastases to theliver (FIG. 26), as compared to the expression in normal colon andliver; and downregulated in 3/3 glioblastoma brain tumors (FIG. 26).

[0357] Human DHDR-4

[0358] The human DHDR-4 gene is highly expressed in liver, kidney,brain, skin, prostate epithelial cells, primary osteoblasts, pituitary,CaCO cells, keratinocytes, aortic endothelial cells, fetal kidney, fetallung, mammary epithelium, fetal spleen, fetal liver, umbilical smoothmuscle, RAII Burkitt Lymphoma cells, lung, prostate, K53 red bloodcells, fetal dorsal spinal cord, insulinoma cells, normal breast andovarian epithelia, retina, HMC-1 mast cells, ovarian ascites, d8dendritic cells, megakaryocytes, human mobilized bone morrow, mammarycarcinoma, in melanoma cells, lymph, vein, U937/A70p B cells, A549concells, WT LN Cap testosterone cells, and esophagus. Significantexpression of DHDR-4 was also observed in aorta, breast, liver, lung,small intestine, glial cells, and thymus. Some expression of DHDR-4 wasobserved in brain, cervix, colon, heart, kidney, muscle, ovary,placenta, testes, and thyroid.

[0359] Human DHDR-4 is also greatly induced in situations of hepatitis Bvirus (HBV) infection (FIG. 28). Human DHDR-4 is expressed at 4-18 foldhigher levels in HBV-infected liver than in normal liver. Theupregulation in HBV infected liver is specific to HBV; no upregulationof human DHDR-4 is seen in hepatitis C virus (HCV) infected liver (FIG.28). Human DHDR-4 expression levels are 12-25 fold higher inHBV-expressing HepG2.2.15 cells than in HepG2 control cells (FIG. 28).Treatment of HepG2 cells with Bayer compound IC50 or IC100 results in astrong induction of human DHDR-4 expression, while treatment ofHBV-expressing HepG2.2.15 cells with Bayer compound IC50 or IC100resultsin a strong decrease in expression of human DHDR-4. Transfection of theHBV X transcription factor alone can induce a 5-fold increase in DHDR-4expression (FIG. 28). In situ hybridization analysis also revealed thathuman DHDR-4 is expressed at a much higher level in HBV positive liverthan in normal liver.

[0360] The human DHDR-4 gene is expressed in a number of tumorigeniccell lines, including MCF-7 (breast tumor), ZR75 (breast tumor), T47D(breast tumor), MDA 231 (breast tumor), MDA 435 (breast tumor), DLD 1(colon tumor—stage C), SW 480, SW 620 (colon tumor—stage C), HCT 116(colon tumor), HT 29 (colon tumor), Colo 205, NCIH 125, NCIH 67, NCIH322, and A549.

[0361] Expression of human DHDR-4 is downregulated in 6/6 glioblastomabrain tumors (FIG. 29), as compared to normal brain. Expression of humanDHDR-4 is downregulated in 6/6 breast tumors, as compared to normalbreast (FIG. 30).

[0362] Expression of human DHDR-4 is upregulated in 6/8 lung tumors, ascompared to normal lung (FIG. 30). In situ data confirmed that humanDHDR-4 is upregulated in poorly differentiated non-small cell carcinomaof the lung (PD NSCCL).

[0363] Expression of human DHDR-4 is downregulated in synchronized A549cells at the time point t=6 after entering the cell cycle, andupregulated at t=12 (FIG. 30). The time point t=0 signifies the G2/Mborder.

Example 2

[0364] Expression of Recombinant DHDR Protein in Bacterial Cells

[0365] In this example, DHDR is expressed as a recombinantglutathione-S-transferase (GST) fusion polypeptide in E. coli and thefusion polypeptide is isolated and characterized. Specifically, DHDR isfused to GST and this fusion polypeptide is expressed in E. coli, e.g.,strain PEB199. Expression of the GST-DHDR fusion protein in PEB199 isinduced with IPTG. The recombinant fusion polypeptide is purified fromcrude bacterial lysates of the induced PEB199 strain by affinitychromatography on glutathione beads. Using polyacrylamide gelelectrophoretic analysis of the polypeptide purified from the bacteriallysates, the molecular weight of the resultant fusion polypeptide isdetermined.

Example 3

[0366] Expression of Recombinant DHDR Protein in COS Cells

[0367] To express the DHDR gene in COS cells, the pcDNA/Amp vector byInvitrogen Corporation (San Diego, Calif.) is used. This vector containsan SV40 origin of replication, an ampicillin resistance gene, an E. colireplication origin, a CMV promoter followed by a polylinker region, andan SV40 intron and polyadenylation site. A DNA fragment encoding theentire DHDR protein and an HA tag (Wilson et al. (1984) Cell 37:767) ora FLAG tag fused in-frame to its 3′ end of the fragment is cloned intothe polylinker region of the vector, thereby placing the expression ofthe recombinant protein under the control of the CMV promoter.

[0368] To construct the plasmid, the DHDR DNA sequence is amplified byPCR using two primers. The 5′ primer contains the restriction site ofinterest followed by approximately twenty nucleotides of the DHDR codingsequence starting from the initiation codon; the 3′ end sequencecontains complementary sequences to the other restriction site ofinterest, a translation stop codon, the HA tag or FLAG tag and the last20 nucleotides of the DHDR coding sequence. The PCR amplified fragmentand the pCDNA/Amp vector are digested with the appropriate restrictionenzymes and the vector is dephosphorylated using the CIAP enzyme (NewEngland Biolabs, Beverly, Mass.). Preferably the two restriction siteschosen are different so that the DHDR gene is inserted in the correctorientation. The ligation mixture is transformed into E. coli cells(strains HB101, DH5α, SURE, available from Stratagene Cloning Systems,La Jolla, Calif., can be used), the transformed culture is plated onampicillin media plates, and resistant colonies are selected. PlasmidDNA is isolated from transformants and examined by restriction analysisfor the presence of the correct fragment.

[0369] COS cells are subsequently transfected with the DHDR-pcDNA/Ampplasmid DNA using the calcium phosphate or calcium chlorideco-precipitation methods, DEAE-dextran-mediated transfection,lipofection, or electroporation. Other suitable methods for transfectinghost cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T.Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989. The expression of the DHDR polypeptide is detected byradiolabeling (³⁵S-methionine or ³⁵S-cysteine available from NEN,Boston, Mass., can be used) and immunoprecipitation (Harlow, E. andLane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1988) using an HA specific monoclonalantibody. Briefly, the cells are labeled for 8 hours with ³⁵S-methionine(or ³⁵S-cysteine). The culture media are then collected and the cellsare lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1%SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culturemedia are precipitated with an HA-specific monoclonal antibody.Precipitated polypeptides are then analyzed by SDS-PAGE.

[0370] Alternatively, DNA containing the DHDR coding sequence is cloneddirectly into the polylinker of the pCDNA/Amp vector using theappropriate restriction sites. The resulting plasmid is transfected intoCOS cells in the manner described above, and the expression of the DHDRpolypeptide is detected by radiolabeling and immunoprecipitation using aDHDR specific monoclonal antibody.

Equivalents

[0371] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

1 16 1 2660 DNA Homo sapiens CDS (63)..(2468) misc_feature (1)..(2660)All occurrences of n = any nucleotide 1 cctttntnrc cacgcgtccg agagcgccccgcagtcttcg cggaaagcgt tcggggtagg 60 cg atg gct gcg acg cgt gca ggg ccccgc gcc cgc gag atc ttc acc 107 Met Ala Ala Thr Arg Ala Gly Pro Arg AlaArg Glu Ile Phe Thr 1 5 10 15 tcg ctg gag tac gga ccg gtg ccg gag agccac gca tgc gca ctg gcc 155 Ser Leu Glu Tyr Gly Pro Val Pro Glu Ser HisAla Cys Ala Leu Ala 20 25 30 tgg ctg gac acc cag gac cgg tgc ttg ggc cactat gtg aat ggg aag 203 Trp Leu Asp Thr Gln Asp Arg Cys Leu Gly His TyrVal Asn Gly Lys 35 40 45 tgg tta aag cct gaa cac aga aat tca gtg cct tgccag gat ccc atc 251 Trp Leu Lys Pro Glu His Arg Asn Ser Val Pro Cys GlnAsp Pro Ile 50 55 60 aca gga gag aac ttg gcc agt tgc ctg cag gca cag gccgag gat gtg 299 Thr Gly Glu Asn Leu Ala Ser Cys Leu Gln Ala Gln Ala GluAsp Val 65 70 75 gct gca gcc gtg gag gca gcc agg atg gca ttt aag ggc tggagt gcg 347 Ala Ala Ala Val Glu Ala Ala Arg Met Ala Phe Lys Gly Trp SerAla 80 85 90 95 cac ccc ggc gtc gtc cgg gcc cag cac ctg acc agg ctg gccgag gtg 395 His Pro Gly Val Val Arg Ala Gln His Leu Thr Arg Leu Ala GluVal 100 105 110 atc cag aag cac cag cgg ctg ctg tgg acc ctg gaa tcc ctggtg act 443 Ile Gln Lys His Gln Arg Leu Leu Trp Thr Leu Glu Ser Leu ValThr 115 120 125 ggg cgg gct gtt cga gag gtt cga gac ggg gac gtc cag ctggcc cag 491 Gly Arg Ala Val Arg Glu Val Arg Asp Gly Asp Val Gln Leu AlaGln 130 135 140 cag ctg ctc cac tac cat gca atc cag gca tcc acc cag gaggag gca 539 Gln Leu Leu His Tyr His Ala Ile Gln Ala Ser Thr Gln Glu GluAla 145 150 155 ctg gca ggc tgg gag ccc atg gga gta att ggc ctc atc ctgcca ccc 587 Leu Ala Gly Trp Glu Pro Met Gly Val Ile Gly Leu Ile Leu ProPro 160 165 170 175 aca ttc tcc ttc ctt gag atg atg tgg agg att tgc cctgcc ctg gct 635 Thr Phe Ser Phe Leu Glu Met Met Trp Arg Ile Cys Pro AlaLeu Ala 180 185 190 gtg ggc tgc acc gtg gtg gcc ctc gtg ccc ccg gcc tccccg gcg ccc 683 Val Gly Cys Thr Val Val Ala Leu Val Pro Pro Ala Ser ProAla Pro 195 200 205 ctc ctc ctg gcc cag ctg gcg ggg gag ctg ggc ccc ttcccg gga atc 731 Leu Leu Leu Ala Gln Leu Ala Gly Glu Leu Gly Pro Phe ProGly Ile 210 215 220 ctg aat gtc gtc agt ggc cct gcg tcc ctg gtg ccc atcctg gcc tcc 779 Leu Asn Val Val Ser Gly Pro Ala Ser Leu Val Pro Ile LeuAla Ser 225 230 235 cag cct gga atc cgg aag gtg gcc ttc tgc gga gcc ccggag gaa ggg 827 Gln Pro Gly Ile Arg Lys Val Ala Phe Cys Gly Ala Pro GluGlu Gly 240 245 250 255 cgt gcc ctt cga cgg agc ctg gcg gga gag tgt gcggag ctg ggc ctg 875 Arg Ala Leu Arg Arg Ser Leu Ala Gly Glu Cys Ala GluLeu Gly Leu 260 265 270 gcg ctg ggg acg gag tcg ctg ctg ctg ctg acg gacacg gcg gac gta 923 Ala Leu Gly Thr Glu Ser Leu Leu Leu Leu Thr Asp ThrAla Asp Val 275 280 285 gac tcg gcc gtg gag ggt gtc gtg gac gcc gcc tggtcc gac cgc ggc 971 Asp Ser Ala Val Glu Gly Val Val Asp Ala Ala Trp SerAsp Arg Gly 290 295 300 ccg ggt ggc ctc agg ctc ctc atc cag gag tct gtgtgg gat gaa gcc 1019 Pro Gly Gly Leu Arg Leu Leu Ile Gln Glu Ser Val TrpAsp Glu Ala 305 310 315 atg aga cgg ctg cag gag cgg atg ggg cgg ctt cggagt ggc cga ggg 1067 Met Arg Arg Leu Gln Glu Arg Met Gly Arg Leu Arg SerGly Arg Gly 320 325 330 335 ctg gat ggg gcc gtg gac atg ggg gcc cgg ggggct gcc gca tgt gac 1115 Leu Asp Gly Ala Val Asp Met Gly Ala Arg Gly AlaAla Ala Cys Asp 340 345 350 ctg gtc cag cgc ttt gtg cgt gag gcc cag agccag ggt gca cag gtg 1163 Leu Val Gln Arg Phe Val Arg Glu Ala Gln Ser GlnGly Ala Gln Val 355 360 365 ttc cag gct ggt gat gtg cct tcg gaa cgc ccattc tat ccc cca acc 1211 Phe Gln Ala Gly Asp Val Pro Ser Glu Arg Pro PheTyr Pro Pro Thr 370 375 380 ttg gtc tcc aac ctg ccc cca gcc tcc cca tgtgcc cag gtg gag gtg 1259 Leu Val Ser Asn Leu Pro Pro Ala Ser Pro Cys AlaGln Val Glu Val 385 390 395 ccg tgg cct gtg gtc gtg gcc tcc ccc ttc cgcaca gcc aag gag gca 1307 Pro Trp Pro Val Val Val Ala Ser Pro Phe Arg ThrAla Lys Glu Ala 400 405 410 415 ctg ttg gtg gcc aac ggg acg ccc cgc gggggc agc gcc agt gtg tgg 1355 Leu Leu Val Ala Asn Gly Thr Pro Arg Gly GlySer Ala Ser Val Trp 420 425 430 agc gag agg ctg ggg cag gcg ctg gag ctgggc tat ggg ctc cag gtg 1403 Ser Glu Arg Leu Gly Gln Ala Leu Glu Leu GlyTyr Gly Leu Gln Val 435 440 445 ggc act gtc tgg atc aac gcc cac ggc ctcaga gac cct tcg gtg ccc 1451 Gly Thr Val Trp Ile Asn Ala His Gly Leu ArgAsp Pro Ser Val Pro 450 455 460 aca ggc ggc tgc aag gag agt ggg tgt tcctgg cac ggg ggc cca gac 1499 Thr Gly Gly Cys Lys Glu Ser Gly Cys Ser TrpHis Gly Gly Pro Asp 465 470 475 ggg ctg tat gag tat ctg cgg ccc tca gggacc cct gcc cgg ctg tcc 1547 Gly Leu Tyr Glu Tyr Leu Arg Pro Ser Gly ThrPro Ala Arg Leu Ser 480 485 490 495 tgc ctc tcc aag aac ctg aac tat gacacc ttt ggc ctc gct gtg ccc 1595 Cys Leu Ser Lys Asn Leu Asn Tyr Asp ThrPhe Gly Leu Ala Val Pro 500 505 510 tca acc ctg ccg gct ggg cct gaa ataggg ccc agc cca gca ccc ccc 1643 Ser Thr Leu Pro Ala Gly Pro Glu Ile GlyPro Ser Pro Ala Pro Pro 515 520 525 tat ggg ctc ttc gtt ggg ggc cgt ttccag gct cct ggg gcc cga agc 1691 Tyr Gly Leu Phe Val Gly Gly Arg Phe GlnAla Pro Gly Ala Arg Ser 530 535 540 tcc agg ccc atc cgg gat tcg tct ggcaat ctc cat ggc tac gtg gct 1739 Ser Arg Pro Ile Arg Asp Ser Ser Gly AsnLeu His Gly Tyr Val Ala 545 550 555 gag ggt gga gcc aag gac atc cga ggtgct gtg gag gcc gct cac cag 1787 Glu Gly Gly Ala Lys Asp Ile Arg Gly AlaVal Glu Ala Ala His Gln 560 565 570 575 gct ttc cct ggc tgg gcg ggc cagtcc cca gga gcc cgg gca gcc ctg 1835 Ala Phe Pro Gly Trp Ala Gly Gln SerPro Gly Ala Arg Ala Ala Leu 580 585 590 ctg tgg gcc ctg gcg gct gca ctggag cgc cgg aag tct acc ctg gcc 1883 Leu Trp Ala Leu Ala Ala Ala Leu GluArg Arg Lys Ser Thr Leu Ala 595 600 605 tca agg ctg gag agg cag gga gcggag ctc aag gct gcg gag gcg gag 1931 Ser Arg Leu Glu Arg Gln Gly Ala GluLeu Lys Ala Ala Glu Ala Glu 610 615 620 gtg gag ctg agc gca aga cga cttcgg gcg tgg ggg gcc cgg gtg cag 1979 Val Glu Leu Ser Ala Arg Arg Leu ArgAla Trp Gly Ala Arg Val Gln 625 630 635 gcc caa ggc cac acc ctg cag gtagcc ggg ctg aga ggc cct gtg ctg 2027 Ala Gln Gly His Thr Leu Gln Val AlaGly Leu Arg Gly Pro Val Leu 640 645 650 655 cgc ctg cgg gag ccg ctg ggtgtg ctg gct gtg gtg tgt ccg gac gag 2075 Arg Leu Arg Glu Pro Leu Gly ValLeu Ala Val Val Cys Pro Asp Glu 660 665 670 tgg ccc ctg ctt gcc ttc gtgtcc ctg ctg gct ccc gcc ctg gcc tac 2123 Trp Pro Leu Leu Ala Phe Val SerLeu Leu Ala Pro Ala Leu Ala Tyr 675 680 685 ggc aac act gtg gtc atg gtgccc agt gcg gcc tgt cct ctg ctg gcc 2171 Gly Asn Thr Val Val Met Val ProSer Ala Ala Cys Pro Leu Leu Ala 690 695 700 ctg gag gtc tgc cag gac atggcc acc gtg ttc cca gca ggc ctg gcc 2219 Leu Glu Val Cys Gln Asp Met AlaThr Val Phe Pro Ala Gly Leu Ala 705 710 715 aac gtg gtg aca gga gac cgggac cat ctg acc cgc tgc ctg gcc ttg 2267 Asn Val Val Thr Gly Asp Arg AspHis Leu Thr Arg Cys Leu Ala Leu 720 725 730 735 cac caa gac gtc cag gccatg tgg tat ttc gga tca gcc cag ggt tcc 2315 His Gln Asp Val Gln Ala MetTrp Tyr Phe Gly Ser Ala Gln Gly Ser 740 745 750 cag ttt gtc gag tgg gcctcg gca gga aac ctc aaa ccg gtg tgg gcg 2363 Gln Phe Val Glu Trp Ala SerAla Gly Asn Leu Lys Pro Val Trp Ala 755 760 765 agc agg ggc tgc ccg cgggcc tgg gac cag gag gcc gag ggg gca ggc 2411 Ser Arg Gly Cys Pro Arg AlaTrp Asp Gln Glu Ala Glu Gly Ala Gly 770 775 780 cca gag ctg ggg ctg cgagtg gcg cgg acc aag gcc ctg tgg ctg cct 2459 Pro Glu Leu Gly Leu Arg ValAla Arg Thr Lys Ala Leu Trp Leu Pro 785 790 795 atg ggg gac tgatgcctgagcgccaccta ctgcattttg gacacctcac 2508 Met Gly Asp 800 accaaggggagatgcacccc acagacacct gggactttcc ccttctggtt cctgtgtctc 2568 ccaataaactctctgaccaa ccctaaaaaa aaaaaaaaaa aaaaaaaaaa rwarmaactt 2628 ctggcagatatgaggctttt ttcttttttt tt 2660 2 802 PRT Homo sapiens 2 Met Ala Ala ThrArg Ala Gly Pro Arg Ala Arg Glu Ile Phe Thr Ser 1 5 10 15 Leu Glu TyrGly Pro Val Pro Glu Ser His Ala Cys Ala Leu Ala Trp 20 25 30 Leu Asp ThrGln Asp Arg Cys Leu Gly His Tyr Val Asn Gly Lys Trp 35 40 45 Leu Lys ProGlu His Arg Asn Ser Val Pro Cys Gln Asp Pro Ile Thr 50 55 60 Gly Glu AsnLeu Ala Ser Cys Leu Gln Ala Gln Ala Glu Asp Val Ala 65 70 75 80 Ala AlaVal Glu Ala Ala Arg Met Ala Phe Lys Gly Trp Ser Ala His 85 90 95 Pro GlyVal Val Arg Ala Gln His Leu Thr Arg Leu Ala Glu Val Ile 100 105 110 GlnLys His Gln Arg Leu Leu Trp Thr Leu Glu Ser Leu Val Thr Gly 115 120 125Arg Ala Val Arg Glu Val Arg Asp Gly Asp Val Gln Leu Ala Gln Gln 130 135140 Leu Leu His Tyr His Ala Ile Gln Ala Ser Thr Gln Glu Glu Ala Leu 145150 155 160 Ala Gly Trp Glu Pro Met Gly Val Ile Gly Leu Ile Leu Pro ProThr 165 170 175 Phe Ser Phe Leu Glu Met Met Trp Arg Ile Cys Pro Ala LeuAla Val 180 185 190 Gly Cys Thr Val Val Ala Leu Val Pro Pro Ala Ser ProAla Pro Leu 195 200 205 Leu Leu Ala Gln Leu Ala Gly Glu Leu Gly Pro PhePro Gly Ile Leu 210 215 220 Asn Val Val Ser Gly Pro Ala Ser Leu Val ProIle Leu Ala Ser Gln 225 230 235 240 Pro Gly Ile Arg Lys Val Ala Phe CysGly Ala Pro Glu Glu Gly Arg 245 250 255 Ala Leu Arg Arg Ser Leu Ala GlyGlu Cys Ala Glu Leu Gly Leu Ala 260 265 270 Leu Gly Thr Glu Ser Leu LeuLeu Leu Thr Asp Thr Ala Asp Val Asp 275 280 285 Ser Ala Val Glu Gly ValVal Asp Ala Ala Trp Ser Asp Arg Gly Pro 290 295 300 Gly Gly Leu Arg LeuLeu Ile Gln Glu Ser Val Trp Asp Glu Ala Met 305 310 315 320 Arg Arg LeuGln Glu Arg Met Gly Arg Leu Arg Ser Gly Arg Gly Leu 325 330 335 Asp GlyAla Val Asp Met Gly Ala Arg Gly Ala Ala Ala Cys Asp Leu 340 345 350 ValGln Arg Phe Val Arg Glu Ala Gln Ser Gln Gly Ala Gln Val Phe 355 360 365Gln Ala Gly Asp Val Pro Ser Glu Arg Pro Phe Tyr Pro Pro Thr Leu 370 375380 Val Ser Asn Leu Pro Pro Ala Ser Pro Cys Ala Gln Val Glu Val Pro 385390 395 400 Trp Pro Val Val Val Ala Ser Pro Phe Arg Thr Ala Lys Glu AlaLeu 405 410 415 Leu Val Ala Asn Gly Thr Pro Arg Gly Gly Ser Ala Ser ValTrp Ser 420 425 430 Glu Arg Leu Gly Gln Ala Leu Glu Leu Gly Tyr Gly LeuGln Val Gly 435 440 445 Thr Val Trp Ile Asn Ala His Gly Leu Arg Asp ProSer Val Pro Thr 450 455 460 Gly Gly Cys Lys Glu Ser Gly Cys Ser Trp HisGly Gly Pro Asp Gly 465 470 475 480 Leu Tyr Glu Tyr Leu Arg Pro Ser GlyThr Pro Ala Arg Leu Ser Cys 485 490 495 Leu Ser Lys Asn Leu Asn Tyr AspThr Phe Gly Leu Ala Val Pro Ser 500 505 510 Thr Leu Pro Ala Gly Pro GluIle Gly Pro Ser Pro Ala Pro Pro Tyr 515 520 525 Gly Leu Phe Val Gly GlyArg Phe Gln Ala Pro Gly Ala Arg Ser Ser 530 535 540 Arg Pro Ile Arg AspSer Ser Gly Asn Leu His Gly Tyr Val Ala Glu 545 550 555 560 Gly Gly AlaLys Asp Ile Arg Gly Ala Val Glu Ala Ala His Gln Ala 565 570 575 Phe ProGly Trp Ala Gly Gln Ser Pro Gly Ala Arg Ala Ala Leu Leu 580 585 590 TrpAla Leu Ala Ala Ala Leu Glu Arg Arg Lys Ser Thr Leu Ala Ser 595 600 605Arg Leu Glu Arg Gln Gly Ala Glu Leu Lys Ala Ala Glu Ala Glu Val 610 615620 Glu Leu Ser Ala Arg Arg Leu Arg Ala Trp Gly Ala Arg Val Gln Ala 625630 635 640 Gln Gly His Thr Leu Gln Val Ala Gly Leu Arg Gly Pro Val LeuArg 645 650 655 Leu Arg Glu Pro Leu Gly Val Leu Ala Val Val Cys Pro AspGlu Trp 660 665 670 Pro Leu Leu Ala Phe Val Ser Leu Leu Ala Pro Ala LeuAla Tyr Gly 675 680 685 Asn Thr Val Val Met Val Pro Ser Ala Ala Cys ProLeu Leu Ala Leu 690 695 700 Glu Val Cys Gln Asp Met Ala Thr Val Phe ProAla Gly Leu Ala Asn 705 710 715 720 Val Val Thr Gly Asp Arg Asp His LeuThr Arg Cys Leu Ala Leu His 725 730 735 Gln Asp Val Gln Ala Met Trp TyrPhe Gly Ser Ala Gln Gly Ser Gln 740 745 750 Phe Val Glu Trp Ala Ser AlaGly Asn Leu Lys Pro Val Trp Ala Ser 755 760 765 Arg Gly Cys Pro Arg AlaTrp Asp Gln Glu Ala Glu Gly Ala Gly Pro 770 775 780 Glu Leu Gly Leu ArgVal Ala Arg Thr Lys Ala Leu Trp Leu Pro Met 785 790 795 800 Gly Asp 32406 DNA Homo sapiens CDS (1)..(2406) 3 atg gct gcg acg cgt gca ggg ccccgc gcc cgc gag atc ttc acc tcg 48 Met Ala Ala Thr Arg Ala Gly Pro ArgAla Arg Glu Ile Phe Thr Ser 1 5 10 15 ctg gag tac gga ccg gtg ccg gagagc cac gca tgc gca ctg gcc tgg 96 Leu Glu Tyr Gly Pro Val Pro Glu SerHis Ala Cys Ala Leu Ala Trp 20 25 30 ctg gac acc cag gac cgg tgc ttg ggccac tat gtg aat ggg aag tgg 144 Leu Asp Thr Gln Asp Arg Cys Leu Gly HisTyr Val Asn Gly Lys Trp 35 40 45 tta aag cct gaa cac aga aat tca gtg ccttgc cag gat ccc atc aca 192 Leu Lys Pro Glu His Arg Asn Ser Val Pro CysGln Asp Pro Ile Thr 50 55 60 gga gag aac ttg gcc agt tgc ctg cag gca caggcc gag gat gtg gct 240 Gly Glu Asn Leu Ala Ser Cys Leu Gln Ala Gln AlaGlu Asp Val Ala 65 70 75 80 gca gcc gtg gag gca gcc agg atg gca ttt aagggc tgg agt gcg cac 288 Ala Ala Val Glu Ala Ala Arg Met Ala Phe Lys GlyTrp Ser Ala His 85 90 95 ccc ggc gtc gtc cgg gcc cag cac ctg acc agg ctggcc gag gtg atc 336 Pro Gly Val Val Arg Ala Gln His Leu Thr Arg Leu AlaGlu Val Ile 100 105 110 cag aag cac cag cgg ctg ctg tgg acc ctg gaa tccctg gtg act ggg 384 Gln Lys His Gln Arg Leu Leu Trp Thr Leu Glu Ser LeuVal Thr Gly 115 120 125 cgg gct gtt cga gag gtt cga gac ggg gac gtc cagctg gcc cag cag 432 Arg Ala Val Arg Glu Val Arg Asp Gly Asp Val Gln LeuAla Gln Gln 130 135 140 ctg ctc cac tac cat gca atc cag gca tcc acc caggag gag gca ctg 480 Leu Leu His Tyr His Ala Ile Gln Ala Ser Thr Gln GluGlu Ala Leu 145 150 155 160 gca ggc tgg gag ccc atg gga gta att ggc ctcatc ctg cca ccc aca 528 Ala Gly Trp Glu Pro Met Gly Val Ile Gly Leu IleLeu Pro Pro Thr 165 170 175 ttc tcc ttc ctt gag atg atg tgg agg att tgccct gcc ctg gct gtg 576 Phe Ser Phe Leu Glu Met Met Trp Arg Ile Cys ProAla Leu Ala Val 180 185 190 ggc tgc acc gtg gtg gcc ctc gtg ccc ccg gcctcc ccg gcg ccc ctc 624 Gly Cys Thr Val Val Ala Leu Val Pro Pro Ala SerPro Ala Pro Leu 195 200 205 ctc ctg gcc cag ctg gcg ggg gag ctg ggc cccttc ccg gga atc ctg 672 Leu Leu Ala Gln Leu Ala Gly Glu Leu Gly Pro PhePro Gly Ile Leu 210 215 220 aat gtc gtc agt ggc cct gcg tcc ctg gtg cccatc ctg gcc tcc cag 720 Asn Val Val Ser Gly Pro Ala Ser Leu Val Pro IleLeu Ala Ser Gln 225 230 235 240 cct gga atc cgg aag gtg gcc ttc tgc ggagcc ccg gag gaa ggg cgt 768 Pro Gly Ile Arg Lys Val Ala Phe Cys Gly AlaPro Glu Glu Gly Arg 245 250 255 gcc ctt cga cgg agc ctg gcg gga gag tgtgcg gag ctg ggc ctg gcg 816 Ala Leu Arg Arg Ser Leu Ala Gly Glu Cys AlaGlu Leu Gly Leu Ala 260 265 270 ctg ggg acg gag tcg ctg ctg ctg ctg acggac acg gcg gac gta gac 864 Leu Gly Thr Glu Ser Leu Leu Leu Leu Thr AspThr Ala Asp Val Asp 275 280 285 tcg gcc gtg gag ggt gtc gtg gac gcc gcctgg tcc gac cgc ggc ccg 912 Ser Ala Val Glu Gly Val Val Asp Ala Ala TrpSer Asp Arg Gly Pro 290 295 300 ggt ggc ctc agg ctc ctc atc cag gag tctgtg tgg gat gaa gcc atg 960 Gly Gly Leu Arg Leu Leu Ile Gln Glu Ser ValTrp Asp Glu Ala Met 305 310 315 320 aga cgg ctg cag gag cgg atg ggg cggctt cgg agt ggc cga ggg ctg 1008 Arg Arg Leu Gln Glu Arg Met Gly Arg LeuArg Ser Gly Arg Gly Leu 325 330 335 gat ggg gcc gtg gac atg ggg gcc cggggg gct gcc gca tgt gac ctg 1056 Asp Gly Ala Val Asp Met Gly Ala Arg GlyAla Ala Ala Cys Asp Leu 340 345 350 gtc cag cgc ttt gtg cgt gag gcc cagagc cag ggt gca cag gtg ttc 1104 Val Gln Arg Phe Val Arg Glu Ala Gln SerGln Gly Ala Gln Val Phe 355 360 365 cag gct ggt gat gtg cct tcg gaa cgccca ttc tat ccc cca acc ttg 1152 Gln Ala Gly Asp Val Pro Ser Glu Arg ProPhe Tyr Pro Pro Thr Leu 370 375 380 gtc tcc aac ctg ccc cca gcc tcc ccatgt gcc cag gtg gag gtg ccg 1200 Val Ser Asn Leu Pro Pro Ala Ser Pro CysAla Gln Val Glu Val Pro 385 390 395 400 tgg cct gtg gtc gtg gcc tcc cccttc cgc aca gcc aag gag gca ctg 1248 Trp Pro Val Val Val Ala Ser Pro PheArg Thr Ala Lys Glu Ala Leu 405 410 415 ttg gtg gcc aac ggg acg ccc cgcggg ggc agc gcc agt gtg tgg agc 1296 Leu Val Ala Asn Gly Thr Pro Arg GlyGly Ser Ala Ser Val Trp Ser 420 425 430 gag agg ctg ggg cag gcg ctg gagctg ggc tat ggg ctc cag gtg ggc 1344 Glu Arg Leu Gly Gln Ala Leu Glu LeuGly Tyr Gly Leu Gln Val Gly 435 440 445 act gtc tgg atc aac gcc cac ggcctc aga gac cct tcg gtg ccc aca 1392 Thr Val Trp Ile Asn Ala His Gly LeuArg Asp Pro Ser Val Pro Thr 450 455 460 ggc ggc tgc aag gag agt ggg tgttcc tgg cac ggg ggc cca gac ggg 1440 Gly Gly Cys Lys Glu Ser Gly Cys SerTrp His Gly Gly Pro Asp Gly 465 470 475 480 ctg tat gag tat ctg cgg ccctca ggg acc cct gcc cgg ctg tcc tgc 1488 Leu Tyr Glu Tyr Leu Arg Pro SerGly Thr Pro Ala Arg Leu Ser Cys 485 490 495 ctc tcc aag aac ctg aac tatgac acc ttt ggc ctc gct gtg ccc tca 1536 Leu Ser Lys Asn Leu Asn Tyr AspThr Phe Gly Leu Ala Val Pro Ser 500 505 510 acc ctg ccg gct ggg cct gaaata ggg ccc agc cca gca ccc ccc tat 1584 Thr Leu Pro Ala Gly Pro Glu IleGly Pro Ser Pro Ala Pro Pro Tyr 515 520 525 ggg ctc ttc gtt ggg ggc cgtttc cag gct cct ggg gcc cga agc tcc 1632 Gly Leu Phe Val Gly Gly Arg PheGln Ala Pro Gly Ala Arg Ser Ser 530 535 540 agg ccc atc cgg gat tcg tctggc aat ctc cat ggc tac gtg gct gag 1680 Arg Pro Ile Arg Asp Ser Ser GlyAsn Leu His Gly Tyr Val Ala Glu 545 550 555 560 ggt gga gcc aag gac atccga ggt gct gtg gag gcc gct cac cag gct 1728 Gly Gly Ala Lys Asp Ile ArgGly Ala Val Glu Ala Ala His Gln Ala 565 570 575 ttc cct ggc tgg gcg ggccag tcc cca gga gcc cgg gca gcc ctg ctg 1776 Phe Pro Gly Trp Ala Gly GlnSer Pro Gly Ala Arg Ala Ala Leu Leu 580 585 590 tgg gcc ctg gcg gct gcactg gag cgc cgg aag tct acc ctg gcc tca 1824 Trp Ala Leu Ala Ala Ala LeuGlu Arg Arg Lys Ser Thr Leu Ala Ser 595 600 605 agg ctg gag agg cag ggagcg gag ctc aag gct gcg gag gcg gag gtg 1872 Arg Leu Glu Arg Gln Gly AlaGlu Leu Lys Ala Ala Glu Ala Glu Val 610 615 620 gag ctg agc gca aga cgactt cgg gcg tgg ggg gcc cgg gtg cag gcc 1920 Glu Leu Ser Ala Arg Arg LeuArg Ala Trp Gly Ala Arg Val Gln Ala 625 630 635 640 caa ggc cac acc ctgcag gta gcc ggg ctg aga ggc cct gtg ctg cgc 1968 Gln Gly His Thr Leu GlnVal Ala Gly Leu Arg Gly Pro Val Leu Arg 645 650 655 ctg cgg gag ccg ctgggt gtg ctg gct gtg gtg tgt ccg gac gag tgg 2016 Leu Arg Glu Pro Leu GlyVal Leu Ala Val Val Cys Pro Asp Glu Trp 660 665 670 ccc ctg ctt gcc ttcgtg tcc ctg ctg gct ccc gcc ctg gcc tac ggc 2064 Pro Leu Leu Ala Phe ValSer Leu Leu Ala Pro Ala Leu Ala Tyr Gly 675 680 685 aac act gtg gtc atggtg ccc agt gcg gcc tgt cct ctg ctg gcc ctg 2112 Asn Thr Val Val Met ValPro Ser Ala Ala Cys Pro Leu Leu Ala Leu 690 695 700 gag gtc tgc cag gacatg gcc acc gtg ttc cca gca ggc ctg gcc aac 2160 Glu Val Cys Gln Asp MetAla Thr Val Phe Pro Ala Gly Leu Ala Asn 705 710 715 720 gtg gtg aca ggagac cgg gac cat ctg acc cgc tgc ctg gcc ttg cac 2208 Val Val Thr Gly AspArg Asp His Leu Thr Arg Cys Leu Ala Leu His 725 730 735 caa gac gtc caggcc atg tgg tat ttc gga tca gcc cag ggt tcc cag 2256 Gln Asp Val Gln AlaMet Trp Tyr Phe Gly Ser Ala Gln Gly Ser Gln 740 745 750 ttt gtc gag tgggcc tcg gca gga aac ctc aaa ccg gtg tgg gcg agc 2304 Phe Val Glu Trp AlaSer Ala Gly Asn Leu Lys Pro Val Trp Ala Ser 755 760 765 agg ggc tgc ccgcgg gcc tgg gac cag gag gcc gag ggg gca ggc cca 2352 Arg Gly Cys Pro ArgAla Trp Asp Gln Glu Ala Glu Gly Ala Gly Pro 770 775 780 gag ctg ggg ctgcga gtg gcg cgg acc aag gcc ctg tgg ctg cct atg 2400 Glu Leu Gly Leu ArgVal Ala Arg Thr Lys Ala Leu Trp Leu Pro Met 785 790 795 800 ggg gac 2406Gly Asp 4 1379 DNA Homo sapiens CDS (331)..(1263) misc_feature(1)..(1379) All occurrences of n = any nucleotide 4 tttggccctcgaggccaaga attcggcacg aggagcaagt ggccttaaca catggatttt 60 cttccaaaaatgcagaccca ttttaattaa gtttgtaatt aaccactggg gagggcaggc 120 cccctggattcggtctgctt tcggagacac tgtgagtaac ttcctatttg ttgaacattt 180 ggggattagcacgcccactg ggtgttcagc ttggaggctt gcacagagct gagctccctg 240 cagccttgggcctccccctg ccctgggagt cctgatcagc gtctctttgc aaagccaatc 300 cccttttactccgttgtccc ccagaacaag atg gga gtc atg gcc atg ctg atg 354 Met Gly ValMet Ala Met Leu Met 1 5 ctc ccc ctg ctg ctg ctg gga atc agc ggc ctc ctcttc att tac caa 402 Leu Pro Leu Leu Leu Leu Gly Ile Ser Gly Leu Leu PheIle Tyr Gln 10 15 20 gag gtg tcc agg ctg tgg tca aag tca gct gtg cag aacaaa gtg gtg 450 Glu Val Ser Arg Leu Trp Ser Lys Ser Ala Val Gln Asn LysVal Val 25 30 35 40 gtg atc acc gat gcc atc tca gga ctg ggc aag gag tgtgct cgg gtg 498 Val Ile Thr Asp Ala Ile Ser Gly Leu Gly Lys Glu Cys AlaArg Val 45 50 55 ttc cac aca ggt ggg gca agg ctg gtg ctg tgt gga aag aactgg gag 546 Phe His Thr Gly Gly Ala Arg Leu Val Leu Cys Gly Lys Asn TrpGlu 60 65 70 agg cta gag aac cta tat gat gcc ttg atc agc gtg gct gac cccagc 594 Arg Leu Glu Asn Leu Tyr Asp Ala Leu Ile Ser Val Ala Asp Pro Ser75 80 85 aag aca ttc acc cca aag ctg gtc ctg ttg gac ctc tca gac atc agc642 Lys Thr Phe Thr Pro Lys Leu Val Leu Leu Asp Leu Ser Asp Ile Ser 9095 100 tgt gtc cca gat gtg gca aaa gaa gtc ctg gat tgc tat ggc tgt gtg690 Cys Val Pro Asp Val Ala Lys Glu Val Leu Asp Cys Tyr Gly Cys Val 105110 115 120 gac atc ctc atc aac aat gcc agt gtg aag gtg aag ggg cct gcccat 738 Asp Ile Leu Ile Asn Asn Ala Ser Val Lys Val Lys Gly Pro Ala His125 130 135 aag att tct ctg gag ctc gac aaa aag atc atg gat gcc aat tacttt 786 Lys Ile Ser Leu Glu Leu Asp Lys Lys Ile Met Asp Ala Asn Tyr Phe140 145 150 ggc ccc atc aca ttg acg aaa gcc ctg ctt ccc aac atg atc tcccgg 834 Gly Pro Ile Thr Leu Thr Lys Ala Leu Leu Pro Asn Met Ile Ser Arg155 160 165 aga aca ggc caa atc gtg tta gtg aat aat atc caa ggg aag tttgga 882 Arg Thr Gly Gln Ile Val Leu Val Asn Asn Ile Gln Gly Lys Phe Gly170 175 180 atc ccg ttc cgt acg act tac gct gcc tcc aag cac gca gcc ctgggc 930 Ile Pro Phe Arg Thr Thr Tyr Ala Ala Ser Lys His Ala Ala Leu Gly185 190 195 200 ttc ttt gac tgc ctc cga gcc gaa gtg gag gaa tac gat gttgtc atc 978 Phe Phe Asp Cys Leu Arg Ala Glu Val Glu Glu Tyr Asp Val ValIle 205 210 215 agc acc gtg agc ccg act ttc atc cgg tcg tac cac gtg tatcca gag 1026 Ser Thr Val Ser Pro Thr Phe Ile Arg Ser Tyr His Val Tyr ProGlu 220 225 230 caa gga aac tgg gaa gct tcc att tgg aaa ttc ttt ttc aggaag ctg 1074 Gln Gly Asn Trp Glu Ala Ser Ile Trp Lys Phe Phe Phe Arg LysLeu 235 240 245 acc tac ggc gtg cac cca gta gag gtg gcg gag gag gtg atgcgc acc 1122 Thr Tyr Gly Val His Pro Val Glu Val Ala Glu Glu Val Met ArgThr 250 255 260 gtg cgg agg aag aag caa gag gtg ttt atg gcc aac ccc atcccc aag 1170 Val Arg Arg Lys Lys Gln Glu Val Phe Met Ala Asn Pro Ile ProLys 265 270 275 280 gcc gcc gtg tac gtc cgc acc ttc ttc ccg gag ttc tttttc gcc gtg 1218 Ala Ala Val Tyr Val Arg Thr Phe Phe Pro Glu Phe Phe PheAla Val 285 290 295 gtg gcc tgt ggg gtg aag gag aag ctc aat gtc ccg gaggag ggg 1263 Val Ala Cys Gly Val Lys Glu Lys Leu Asn Val Pro Glu Glu Gly300 305 310 taactgcagg aggccaaatg ggccacccct tggaaataaa ggtttttctggcaaaaaaaa 1323 aaaaaaaaaa aaantttgcg gccgcaagct tattcccttt agggagggttaatttt 1379 5 311 PRT Homo sapiens 5 Met Gly Val Met Ala Met Leu Met LeuPro Leu Leu Leu Leu Gly Ile 1 5 10 15 Ser Gly Leu Leu Phe Ile Tyr GlnGlu Val Ser Arg Leu Trp Ser Lys 20 25 30 Ser Ala Val Gln Asn Lys Val ValVal Ile Thr Asp Ala Ile Ser Gly 35 40 45 Leu Gly Lys Glu Cys Ala Arg ValPhe His Thr Gly Gly Ala Arg Leu 50 55 60 Val Leu Cys Gly Lys Asn Trp GluArg Leu Glu Asn Leu Tyr Asp Ala 65 70 75 80 Leu Ile Ser Val Ala Asp ProSer Lys Thr Phe Thr Pro Lys Leu Val 85 90 95 Leu Leu Asp Leu Ser Asp IleSer Cys Val Pro Asp Val Ala Lys Glu 100 105 110 Val Leu Asp Cys Tyr GlyCys Val Asp Ile Leu Ile Asn Asn Ala Ser 115 120 125 Val Lys Val Lys GlyPro Ala His Lys Ile Ser Leu Glu Leu Asp Lys 130 135 140 Lys Ile Met AspAla Asn Tyr Phe Gly Pro Ile Thr Leu Thr Lys Ala 145 150 155 160 Leu LeuPro Asn Met Ile Ser Arg Arg Thr Gly Gln Ile Val Leu Val 165 170 175 AsnAsn Ile Gln Gly Lys Phe Gly Ile Pro Phe Arg Thr Thr Tyr Ala 180 185 190Ala Ser Lys His Ala Ala Leu Gly Phe Phe Asp Cys Leu Arg Ala Glu 195 200205 Val Glu Glu Tyr Asp Val Val Ile Ser Thr Val Ser Pro Thr Phe Ile 210215 220 Arg Ser Tyr His Val Tyr Pro Glu Gln Gly Asn Trp Glu Ala Ser Ile225 230 235 240 Trp Lys Phe Phe Phe Arg Lys Leu Thr Tyr Gly Val His ProVal Glu 245 250 255 Val Ala Glu Glu Val Met Arg Thr Val Arg Arg Lys LysGln Glu Val 260 265 270 Phe Met Ala Asn Pro Ile Pro Lys Ala Ala Val TyrVal Arg Thr Phe 275 280 285 Phe Pro Glu Phe Phe Phe Ala Val Val Ala CysGly Val Lys Glu Lys 290 295 300 Leu Asn Val Pro Glu Glu Gly 305 310 6933 DNA Homo sapiens CDS (1)..(933) 6 atg gga gtc atg gcc atg ctg atgctc ccc ctg ctg ctg ctg gga atc 48 Met Gly Val Met Ala Met Leu Met LeuPro Leu Leu Leu Leu Gly Ile 1 5 10 15 agc ggc ctc ctc ttc att tac caagag gtg tcc agg ctg tgg tca aag 96 Ser Gly Leu Leu Phe Ile Tyr Gln GluVal Ser Arg Leu Trp Ser Lys 20 25 30 tca gct gtg cag aac aaa gtg gtg gtgatc acc gat gcc atc tca gga 144 Ser Ala Val Gln Asn Lys Val Val Val IleThr Asp Ala Ile Ser Gly 35 40 45 ctg ggc aag gag tgt gct cgg gtg ttc cacaca ggt ggg gca agg ctg 192 Leu Gly Lys Glu Cys Ala Arg Val Phe His ThrGly Gly Ala Arg Leu 50 55 60 gtg ctg tgt gga aag aac tgg gag agg cta gagaac cta tat gat gcc 240 Val Leu Cys Gly Lys Asn Trp Glu Arg Leu Glu AsnLeu Tyr Asp Ala 65 70 75 80 ttg atc agc gtg gct gac ccc agc aag aca ttcacc cca aag ctg gtc 288 Leu Ile Ser Val Ala Asp Pro Ser Lys Thr Phe ThrPro Lys Leu Val 85 90 95 ctg ttg gac ctc tca gac atc agc tgt gtc cca gatgtg gca aaa gaa 336 Leu Leu Asp Leu Ser Asp Ile Ser Cys Val Pro Asp ValAla Lys Glu 100 105 110 gtc ctg gat tgc tat ggc tgt gtg gac atc ctc atcaac aat gcc agt 384 Val Leu Asp Cys Tyr Gly Cys Val Asp Ile Leu Ile AsnAsn Ala Ser 115 120 125 gtg aag gtg aag ggg cct gcc cat aag att tct ctggag ctc gac aaa 432 Val Lys Val Lys Gly Pro Ala His Lys Ile Ser Leu GluLeu Asp Lys 130 135 140 aag atc atg gat gcc aat tac ttt ggc ccc atc acattg acg aaa gcc 480 Lys Ile Met Asp Ala Asn Tyr Phe Gly Pro Ile Thr LeuThr Lys Ala 145 150 155 160 ctg ctt ccc aac atg atc tcc cgg aga aca ggccaa atc gtg tta gtg 528 Leu Leu Pro Asn Met Ile Ser Arg Arg Thr Gly GlnIle Val Leu Val 165 170 175 aat aat atc caa ggg aag ttt gga atc ccg ttccgt acg act tac gct 576 Asn Asn Ile Gln Gly Lys Phe Gly Ile Pro Phe ArgThr Thr Tyr Ala 180 185 190 gcc tcc aag cac gca gcc ctg ggc ttc ttt gactgc ctc cga gcc gaa 624 Ala Ser Lys His Ala Ala Leu Gly Phe Phe Asp CysLeu Arg Ala Glu 195 200 205 gtg gag gaa tac gat gtt gtc atc agc acc gtgagc ccg act ttc atc 672 Val Glu Glu Tyr Asp Val Val Ile Ser Thr Val SerPro Thr Phe Ile 210 215 220 cgg tcg tac cac gtg tat cca gag caa gga aactgg gaa gct tcc att 720 Arg Ser Tyr His Val Tyr Pro Glu Gln Gly Asn TrpGlu Ala Ser Ile 225 230 235 240 tgg aaa ttc ttt ttc agg aag ctg acc tacggc gtg cac cca gta gag 768 Trp Lys Phe Phe Phe Arg Lys Leu Thr Tyr GlyVal His Pro Val Glu 245 250 255 gtg gcg gag gag gtg atg cgc acc gtg cggagg aag aag caa gag gtg 816 Val Ala Glu Glu Val Met Arg Thr Val Arg ArgLys Lys Gln Glu Val 260 265 270 ttt atg gcc aac ccc atc ccc aag gcc gccgtg tac gtc cgc acc ttc 864 Phe Met Ala Asn Pro Ile Pro Lys Ala Ala ValTyr Val Arg Thr Phe 275 280 285 ttc ccg gag ttc ttt ttc gcc gtg gtg gcctgt ggg gtg aag gag aag 912 Phe Pro Glu Phe Phe Phe Ala Val Val Ala CysGly Val Lys Glu Lys 290 295 300 ctc aat gtc ccg gag gag ggg 933 Leu AsnVal Pro Glu Glu Gly 305 310 7 1725 DNA Homo sapiens CDS (281)..(1387)misc_feature (1)..(1725) All occurrences of n = any nucleotide 7gagaaggagg agccagcgga aggacggtgt gcgggccggc cagccctgga cgaaagaaga 60gggcccctcc aggccagtct gggcaccctg ggatagcggc tgcagccatc agcaggggca 120gacggcaggt ggcctggttg ctgcagctcc caggatcagc tctgccctcc ccgcaaacgc 180cagcctcgtc accgctccag ggcacctcca gcagtaacag gtggttgcag caggtggcag 240ccagcccctg gatgagccaa ggtctcttcc ccagccaggc atg gcc gac tct gca 295 MetAla Asp Ser Ala 1 5 cag gcc cag aag ctg gtg tac ctg gtc aca ggg ggc tgtggc ttc ctg 343 Gln Ala Gln Lys Leu Val Tyr Leu Val Thr Gly Gly Cys GlyPhe Leu 10 15 20 gga gag cac gtg gtg cga atg ctg ctg cag cgg gag ccc cggctc ggg 391 Gly Glu His Val Val Arg Met Leu Leu Gln Arg Glu Pro Arg LeuGly 25 30 35 gag ctg cgg gtc ttt gac caa cac ctg ggt ccc tgg ctg gag gagctg 439 Glu Leu Arg Val Phe Asp Gln His Leu Gly Pro Trp Leu Glu Glu Leu40 45 50 aag aca ggg cct gtg agg gtg act gcc atc cag ggg gac gtg acc cag487 Lys Thr Gly Pro Val Arg Val Thr Ala Ile Gln Gly Asp Val Thr Gln 5560 65 gcc cat gag gtg gca gca gct gtg gcc gga gcc cat gtg gtc atc cac535 Ala His Glu Val Ala Ala Ala Val Ala Gly Ala His Val Val Ile His 7075 80 85 acg gct ggg ctg gta gac gtg ttt ggc agg gcc agt ccc aag acc atc583 Thr Ala Gly Leu Val Asp Val Phe Gly Arg Ala Ser Pro Lys Thr Ile 9095 100 cat gag gtc aac gtg cag ggt acc cgg aac gtg atc gag gct tgt gtg631 His Glu Val Asn Val Gln Gly Thr Arg Asn Val Ile Glu Ala Cys Val 105110 115 cag acc gga aca cgg ttc ctg gtc tac acc agc agc atg gaa gtt gtg679 Gln Thr Gly Thr Arg Phe Leu Val Tyr Thr Ser Ser Met Glu Val Val 120125 130 ggg cct aac acc aaa ggt cac ccc ttc tac agg ggc aac gaa gac acc727 Gly Pro Asn Thr Lys Gly His Pro Phe Tyr Arg Gly Asn Glu Asp Thr 135140 145 cca tac gaa gca gtg cac agg cac ccc tat cct tgc agc aag gcc ctg775 Pro Tyr Glu Ala Val His Arg His Pro Tyr Pro Cys Ser Lys Ala Leu 150155 160 165 gcc gag tgg ctg gtc ctg gag gcc aac ggg agg aag gtc cgt gggggg 823 Ala Glu Trp Leu Val Leu Glu Ala Asn Gly Arg Lys Val Arg Gly Gly170 175 180 ctg ccc ctg gtg acg tgt gcc ctt cgt ccc acg ggc atc tac ggtgaa 871 Leu Pro Leu Val Thr Cys Ala Leu Arg Pro Thr Gly Ile Tyr Gly Glu185 190 195 ggc cac cag atc atg agg gac ttc tac cgc cag ggc ctg cgc ctggga 919 Gly His Gln Ile Met Arg Asp Phe Tyr Arg Gln Gly Leu Arg Leu Gly200 205 210 ggt tgg ctc ttc cgg gcc atc ccg gcc tct gtg gag cat ggc cgggtc 967 Gly Trp Leu Phe Arg Ala Ile Pro Ala Ser Val Glu His Gly Arg Val215 220 225 tat gtg ggc aat gtt gcc tgg atg cac gtg ctg gca gcc cgg gagctg 1015 Tyr Val Gly Asn Val Ala Trp Met His Val Leu Ala Ala Arg Glu Leu230 235 240 245 gag cag cgg gca gcc ctg atg ggc ggc cag gta tac ttc tgctac gat 1063 Glu Gln Arg Ala Ala Leu Met Gly Gly Gln Val Tyr Phe Cys TyrAsp 250 255 260 gga tca ccc tac agg agc tac gag gat ttc aac atg gag ttcctg ggc 1111 Gly Ser Pro Tyr Arg Ser Tyr Glu Asp Phe Asn Met Glu Phe LeuGly 265 270 275 ccc tgc gga ctg cgg ctg gtg ggc gcc cgc cca ttg ctg ccctac tgg 1159 Pro Cys Gly Leu Arg Leu Val Gly Ala Arg Pro Leu Leu Pro TyrTrp 280 285 290 ctg ctg gtg ttc ctg gct gcc ctc aat gcc ctg ctg cag tggctg ctg 1207 Leu Leu Val Phe Leu Ala Ala Leu Asn Ala Leu Leu Gln Trp LeuLeu 295 300 305 cgg cca ctg gtg ctc tac gca ccc ctg ctg aac ccc tac acgctg gcc 1255 Arg Pro Leu Val Leu Tyr Ala Pro Leu Leu Asn Pro Tyr Thr LeuAla 310 315 320 325 gtg gcc aac acc acc ttc acc gtc agc acc gac aag gctcag cgc cat 1303 Val Ala Asn Thr Thr Phe Thr Val Ser Thr Asp Lys Ala GlnArg His 330 335 340 ttc ggc tat gag ccc ctg ttc tcg tgg gag gat agc cggacc cgc acc 1351 Phe Gly Tyr Glu Pro Leu Phe Ser Trp Glu Asp Ser Arg ThrArg Thr 345 350 355 att ctc tgg gta cag gcc gct acg ggt tca gcc cagtgacggtggg 1397 Ile Leu Trp Val Gln Ala Ala Thr Gly Ser Ala Gln 360 365gctggggcct ggaggcccag atacagcaca tccacccagg tcccgagccc tcacaccctg 1457gacgggaagg gacagctgca ttccagagca ggaggcaggg ctctggggcc agaatggctg 1517tccttgtcgt agagccctcc acattttctt tttctttttt gagacagggt cttgctctgt 1577cacccagact ggaatgcaag tggtgtgant cataagctca ctngmaccct yaanccttct 1637gggttcaagc aatccttnct ngcctyaanc cttctngaac aagcttggga nccacaggtg 1697cacgccancc acancctggc tttttttt 1725 8 369 PRT Homo sapiens 8 Met Ala AspSer Ala Gln Ala Gln Lys Leu Val Tyr Leu Val Thr Gly 1 5 10 15 Gly CysGly Phe Leu Gly Glu His Val Val Arg Met Leu Leu Gln Arg 20 25 30 Glu ProArg Leu Gly Glu Leu Arg Val Phe Asp Gln His Leu Gly Pro 35 40 45 Trp LeuGlu Glu Leu Lys Thr Gly Pro Val Arg Val Thr Ala Ile Gln 50 55 60 Gly AspVal Thr Gln Ala His Glu Val Ala Ala Ala Val Ala Gly Ala 65 70 75 80 HisVal Val Ile His Thr Ala Gly Leu Val Asp Val Phe Gly Arg Ala 85 90 95 SerPro Lys Thr Ile His Glu Val Asn Val Gln Gly Thr Arg Asn Val 100 105 110Ile Glu Ala Cys Val Gln Thr Gly Thr Arg Phe Leu Val Tyr Thr Ser 115 120125 Ser Met Glu Val Val Gly Pro Asn Thr Lys Gly His Pro Phe Tyr Arg 130135 140 Gly Asn Glu Asp Thr Pro Tyr Glu Ala Val His Arg His Pro Tyr Pro145 150 155 160 Cys Ser Lys Ala Leu Ala Glu Trp Leu Val Leu Glu Ala AsnGly Arg 165 170 175 Lys Val Arg Gly Gly Leu Pro Leu Val Thr Cys Ala LeuArg Pro Thr 180 185 190 Gly Ile Tyr Gly Glu Gly His Gln Ile Met Arg AspPhe Tyr Arg Gln 195 200 205 Gly Leu Arg Leu Gly Gly Trp Leu Phe Arg AlaIle Pro Ala Ser Val 210 215 220 Glu His Gly Arg Val Tyr Val Gly Asn ValAla Trp Met His Val Leu 225 230 235 240 Ala Ala Arg Glu Leu Glu Gln ArgAla Ala Leu Met Gly Gly Gln Val 245 250 255 Tyr Phe Cys Tyr Asp Gly SerPro Tyr Arg Ser Tyr Glu Asp Phe Asn 260 265 270 Met Glu Phe Leu Gly ProCys Gly Leu Arg Leu Val Gly Ala Arg Pro 275 280 285 Leu Leu Pro Tyr TrpLeu Leu Val Phe Leu Ala Ala Leu Asn Ala Leu 290 295 300 Leu Gln Trp LeuLeu Arg Pro Leu Val Leu Tyr Ala Pro Leu Leu Asn 305 310 315 320 Pro TyrThr Leu Ala Val Ala Asn Thr Thr Phe Thr Val Ser Thr Asp 325 330 335 LysAla Gln Arg His Phe Gly Tyr Glu Pro Leu Phe Ser Trp Glu Asp 340 345 350Ser Arg Thr Arg Thr Ile Leu Trp Val Gln Ala Ala Thr Gly Ser Ala 355 360365 Gln 9 1107 DNA Homo sapiens CDS (1)..(1107) 9 atg gcc gac tct gcacag gcc cag aag ctg gtg tac ctg gtc aca ggg 48 Met Ala Asp Ser Ala GlnAla Gln Lys Leu Val Tyr Leu Val Thr Gly 1 5 10 15 ggc tgt ggc ttc ctggga gag cac gtg gtg cga atg ctg ctg cag cgg 96 Gly Cys Gly Phe Leu GlyGlu His Val Val Arg Met Leu Leu Gln Arg 20 25 30 gag ccc cgg ctc ggg gagctg cgg gtc ttt gac caa cac ctg ggt ccc 144 Glu Pro Arg Leu Gly Glu LeuArg Val Phe Asp Gln His Leu Gly Pro 35 40 45 tgg ctg gag gag ctg aag acaggg cct gtg agg gtg act gcc atc cag 192 Trp Leu Glu Glu Leu Lys Thr GlyPro Val Arg Val Thr Ala Ile Gln 50 55 60 ggg gac gtg acc cag gcc cat gaggtg gca gca gct gtg gcc gga gcc 240 Gly Asp Val Thr Gln Ala His Glu ValAla Ala Ala Val Ala Gly Ala 65 70 75 80 cat gtg gtc atc cac acg gct gggctg gta gac gtg ttt ggc agg gcc 288 His Val Val Ile His Thr Ala Gly LeuVal Asp Val Phe Gly Arg Ala 85 90 95 agt ccc aag acc atc cat gag gtc aacgtg cag ggt acc cgg aac gtg 336 Ser Pro Lys Thr Ile His Glu Val Asn ValGln Gly Thr Arg Asn Val 100 105 110 atc gag gct tgt gtg cag acc gga acacgg ttc ctg gtc tac acc agc 384 Ile Glu Ala Cys Val Gln Thr Gly Thr ArgPhe Leu Val Tyr Thr Ser 115 120 125 agc atg gaa gtt gtg ggg cct aac accaaa ggt cac ccc ttc tac agg 432 Ser Met Glu Val Val Gly Pro Asn Thr LysGly His Pro Phe Tyr Arg 130 135 140 ggc aac gaa gac acc cca tac gaa gcagtg cac agg cac ccc tat cct 480 Gly Asn Glu Asp Thr Pro Tyr Glu Ala ValHis Arg His Pro Tyr Pro 145 150 155 160 tgc agc aag gcc ctg gcc gag tggctg gtc ctg gag gcc aac ggg agg 528 Cys Ser Lys Ala Leu Ala Glu Trp LeuVal Leu Glu Ala Asn Gly Arg 165 170 175 aag gtc cgt ggg ggg ctg ccc ctggtg acg tgt gcc ctt cgt ccc acg 576 Lys Val Arg Gly Gly Leu Pro Leu ValThr Cys Ala Leu Arg Pro Thr 180 185 190 ggc atc tac ggt gaa ggc cac cagatc atg agg gac ttc tac cgc cag 624 Gly Ile Tyr Gly Glu Gly His Gln IleMet Arg Asp Phe Tyr Arg Gln 195 200 205 ggc ctg cgc ctg gga ggt tgg ctcttc cgg gcc atc ccg gcc tct gtg 672 Gly Leu Arg Leu Gly Gly Trp Leu PheArg Ala Ile Pro Ala Ser Val 210 215 220 gag cat ggc cgg gtc tat gtg ggcaat gtt gcc tgg atg cac gtg ctg 720 Glu His Gly Arg Val Tyr Val Gly AsnVal Ala Trp Met His Val Leu 225 230 235 240 gca gcc cgg gag ctg gag cagcgg gca gcc ctg atg ggc ggc cag gta 768 Ala Ala Arg Glu Leu Glu Gln ArgAla Ala Leu Met Gly Gly Gln Val 245 250 255 tac ttc tgc tac gat gga tcaccc tac agg agc tac gag gat ttc aac 816 Tyr Phe Cys Tyr Asp Gly Ser ProTyr Arg Ser Tyr Glu Asp Phe Asn 260 265 270 atg gag ttc ctg ggc ccc tgcgga ctg cgg ctg gtg ggc gcc cgc cca 864 Met Glu Phe Leu Gly Pro Cys GlyLeu Arg Leu Val Gly Ala Arg Pro 275 280 285 ttg ctg ccc tac tgg ctg ctggtg ttc ctg gct gcc ctc aat gcc ctg 912 Leu Leu Pro Tyr Trp Leu Leu ValPhe Leu Ala Ala Leu Asn Ala Leu 290 295 300 ctg cag tgg ctg ctg cgg ccactg gtg ctc tac gca ccc ctg ctg aac 960 Leu Gln Trp Leu Leu Arg Pro LeuVal Leu Tyr Ala Pro Leu Leu Asn 305 310 315 320 ccc tac acg ctg gcc gtggcc aac acc acc ttc acc gtc agc acc gac 1008 Pro Tyr Thr Leu Ala Val AlaAsn Thr Thr Phe Thr Val Ser Thr Asp 325 330 335 aag gct cag cgc cat ttcggc tat gag ccc ctg ttc tcg tgg gag gat 1056 Lys Ala Gln Arg His Phe GlyTyr Glu Pro Leu Phe Ser Trp Glu Asp 340 345 350 agc cgg acc cgc acc attctc tgg gta cag gcc gct acg ggt tca gcc 1104 Ser Arg Thr Arg Thr Ile LeuTrp Val Gln Ala Ala Thr Gly Ser Ala 355 360 365 cag 1107 Gln 10 1209 DNAHomo sapiens CDS (61)..(1026) 10 cccacgcgtc cgcccacgcg tccgcggacgcgtgggcgga cgcgtgggcg cccgcctcga 60 atg tcc ctg aga ccc aga agg gcc tgcgct cag ctg ctc tgg cac ccc 108 Met Ser Leu Arg Pro Arg Arg Ala Cys AlaGln Leu Leu Trp His Pro 1 5 10 15 gct gca ggg atg gcc tcc tgg gct aagggc agg agc tac ctg gcg cct 156 Ala Ala Gly Met Ala Ser Trp Ala Lys GlyArg Ser Tyr Leu Ala Pro 20 25 30 ggt ttg ctg cag ggc caa gtg gcc atc gtcacc ggc ggg gcc acg ggc 204 Gly Leu Leu Gln Gly Gln Val Ala Ile Val ThrGly Gly Ala Thr Gly 35 40 45 atc gga aaa gcc atc gtg aag gag ctc ctg gagctg ggg agt aat gtg 252 Ile Gly Lys Ala Ile Val Lys Glu Leu Leu Glu LeuGly Ser Asn Val 50 55 60 gtc att gca tcc cgt aag ttg gag aga ttg aag tctgcg gca gat gaa 300 Val Ile Ala Ser Arg Lys Leu Glu Arg Leu Lys Ser AlaAla Asp Glu 65 70 75 80 ctg cag gcc aac cta cct ccc aca aag cag gca cgagtc att ccc ata 348 Leu Gln Ala Asn Leu Pro Pro Thr Lys Gln Ala Arg ValIle Pro Ile 85 90 95 caa tgc aac atc cgg aat gag gag gag gtg aat aat ttggtc aaa tct 396 Gln Cys Asn Ile Arg Asn Glu Glu Glu Val Asn Asn Leu ValLys Ser 100 105 110 acc tta gat act ttt ggt aag atc aat ttc ttg gtg aacaat gga gga 444 Thr Leu Asp Thr Phe Gly Lys Ile Asn Phe Leu Val Asn AsnGly Gly 115 120 125 ggc cag ttt ctt tcc cct gct gaa cac atc agt tct aaggga tgg cac 492 Gly Gln Phe Leu Ser Pro Ala Glu His Ile Ser Ser Lys GlyTrp His 130 135 140 gct gtg ctt gag acc aac ctg acg ggt acc ttc tac atgtgc aaa gca 540 Ala Val Leu Glu Thr Asn Leu Thr Gly Thr Phe Tyr Met CysLys Ala 145 150 155 160 gtt tac agc tcc tgg atg aaa gag cat gga gga tctatc gtc aat atc 588 Val Tyr Ser Ser Trp Met Lys Glu His Gly Gly Ser IleVal Asn Ile 165 170 175 att gtc cct act aaa gct gga ttt cca tta gct gtgcat tct gga gct 636 Ile Val Pro Thr Lys Ala Gly Phe Pro Leu Ala Val HisSer Gly Ala 180 185 190 gca aga gca ggt gtt tac aac ctc acc aaa tct ttagct ttg gaa tgg 684 Ala Arg Ala Gly Val Tyr Asn Leu Thr Lys Ser Leu AlaLeu Glu Trp 195 200 205 gcc tgc agt gga ata cgg atc aat tgt gtt gcc cctgga gtt att tat 732 Ala Cys Ser Gly Ile Arg Ile Asn Cys Val Ala Pro GlyVal Ile Tyr 210 215 220 tcc cag act gct gtg gag aac tat ggt tcc tgg ggacaa agc ttc ttt 780 Ser Gln Thr Ala Val Glu Asn Tyr Gly Ser Trp Gly GlnSer Phe Phe 225 230 235 240 gaa ggg tct ttt cag aaa atc ccc gct aaa cgaatt ggt gtt cct gag 828 Glu Gly Ser Phe Gln Lys Ile Pro Ala Lys Arg IleGly Val Pro Glu 245 250 255 gag gtc tcc tct gtg gtc tgc ttc cta ctg tctcct gca gct tcc ttc 876 Glu Val Ser Ser Val Val Cys Phe Leu Leu Ser ProAla Ala Ser Phe 260 265 270 atc act gga cag tcg gtg gat gtg gat ggg ggccgg agt ctc tat act 924 Ile Thr Gly Gln Ser Val Asp Val Asp Gly Gly ArgSer Leu Tyr Thr 275 280 285 cac tcg tat gag gta cca gat cat gac aac tggccc aag gga gca ggg 972 His Ser Tyr Glu Val Pro Asp His Asp Asn Trp ProLys Gly Ala Gly 290 295 300 gac ctt tct gtt gtc aaa aag atg aag gag acctta aag gag aaa gct 1020 Asp Leu Ser Val Val Lys Lys Met Lys Glu Thr LeuLys Glu Lys Ala 305 310 315 320 aag ctc tgagctgagg aaacaaggtg tcctccatccccagtgcctt cacatcttga 1076 Lys Leu ggatatgctt ctgtactttt taaaagcttatagttggtat ggaaaacatt tttcttattt 1136 ttaagtgtta ttaattatat ctatggaaaaactattcctg aaatatatac agtcttatgt 1196 cccaaaaaaa aaa 1209 11 322 PRTHomo sapiens 11 Met Ser Leu Arg Pro Arg Arg Ala Cys Ala Gln Leu Leu TrpHis Pro 1 5 10 15 Ala Ala Gly Met Ala Ser Trp Ala Lys Gly Arg Ser TyrLeu Ala Pro 20 25 30 Gly Leu Leu Gln Gly Gln Val Ala Ile Val Thr Gly GlyAla Thr Gly 35 40 45 Ile Gly Lys Ala Ile Val Lys Glu Leu Leu Glu Leu GlySer Asn Val 50 55 60 Val Ile Ala Ser Arg Lys Leu Glu Arg Leu Lys Ser AlaAla Asp Glu 65 70 75 80 Leu Gln Ala Asn Leu Pro Pro Thr Lys Gln Ala ArgVal Ile Pro Ile 85 90 95 Gln Cys Asn Ile Arg Asn Glu Glu Glu Val Asn AsnLeu Val Lys Ser 100 105 110 Thr Leu Asp Thr Phe Gly Lys Ile Asn Phe LeuVal Asn Asn Gly Gly 115 120 125 Gly Gln Phe Leu Ser Pro Ala Glu His IleSer Ser Lys Gly Trp His 130 135 140 Ala Val Leu Glu Thr Asn Leu Thr GlyThr Phe Tyr Met Cys Lys Ala 145 150 155 160 Val Tyr Ser Ser Trp Met LysGlu His Gly Gly Ser Ile Val Asn Ile 165 170 175 Ile Val Pro Thr Lys AlaGly Phe Pro Leu Ala Val His Ser Gly Ala 180 185 190 Ala Arg Ala Gly ValTyr Asn Leu Thr Lys Ser Leu Ala Leu Glu Trp 195 200 205 Ala Cys Ser GlyIle Arg Ile Asn Cys Val Ala Pro Gly Val Ile Tyr 210 215 220 Ser Gln ThrAla Val Glu Asn Tyr Gly Ser Trp Gly Gln Ser Phe Phe 225 230 235 240 GluGly Ser Phe Gln Lys Ile Pro Ala Lys Arg Ile Gly Val Pro Glu 245 250 255Glu Val Ser Ser Val Val Cys Phe Leu Leu Ser Pro Ala Ala Ser Phe 260 265270 Ile Thr Gly Gln Ser Val Asp Val Asp Gly Gly Arg Ser Leu Tyr Thr 275280 285 His Ser Tyr Glu Val Pro Asp His Asp Asn Trp Pro Lys Gly Ala Gly290 295 300 Asp Leu Ser Val Val Lys Lys Met Lys Glu Thr Leu Lys Glu LysAla 305 310 315 320 Lys Leu 12 966 DNA Homo sapiens CDS (1)..(966) 12atg tcc ctg aga ccc aga agg gcc tgc gct cag ctg ctc tgg cac ccc 48 MetSer Leu Arg Pro Arg Arg Ala Cys Ala Gln Leu Leu Trp His Pro 1 5 10 15gct gca ggg atg gcc tcc tgg gct aag ggc agg agc tac ctg gcg cct 96 AlaAla Gly Met Ala Ser Trp Ala Lys Gly Arg Ser Tyr Leu Ala Pro 20 25 30 ggtttg ctg cag ggc caa gtg gcc atc gtc acc ggc ggg gcc acg ggc 144 Gly LeuLeu Gln Gly Gln Val Ala Ile Val Thr Gly Gly Ala Thr Gly 35 40 45 atc ggaaaa gcc atc gtg aag gag ctc ctg gag ctg ggg agt aat gtg 192 Ile Gly LysAla Ile Val Lys Glu Leu Leu Glu Leu Gly Ser Asn Val 50 55 60 gtc att gcatcc cgt aag ttg gag aga ttg aag tct gcg gca gat gaa 240 Val Ile Ala SerArg Lys Leu Glu Arg Leu Lys Ser Ala Ala Asp Glu 65 70 75 80 ctg cag gccaac cta cct ccc aca aag cag gca cga gtc att ccc ata 288 Leu Gln Ala AsnLeu Pro Pro Thr Lys Gln Ala Arg Val Ile Pro Ile 85 90 95 caa tgc aac atccgg aat gag gag gag gtg aat aat ttg gtc aaa tct 336 Gln Cys Asn Ile ArgAsn Glu Glu Glu Val Asn Asn Leu Val Lys Ser 100 105 110 acc tta gat actttt ggt aag atc aat ttc ttg gtg aac aat gga gga 384 Thr Leu Asp Thr PheGly Lys Ile Asn Phe Leu Val Asn Asn Gly Gly 115 120 125 ggc cag ttt ctttcc cct gct gaa cac atc agt tct aag gga tgg cac 432 Gly Gln Phe Leu SerPro Ala Glu His Ile Ser Ser Lys Gly Trp His 130 135 140 gct gtg ctt gagacc aac ctg acg ggt acc ttc tac atg tgc aaa gca 480 Ala Val Leu Glu ThrAsn Leu Thr Gly Thr Phe Tyr Met Cys Lys Ala 145 150 155 160 gtt tac agctcc tgg atg aaa gag cat gga gga tct atc gtc aat atc 528 Val Tyr Ser SerTrp Met Lys Glu His Gly Gly Ser Ile Val Asn Ile 165 170 175 att gtc cctact aaa gct gga ttt cca tta gct gtg cat tct gga gct 576 Ile Val Pro ThrLys Ala Gly Phe Pro Leu Ala Val His Ser Gly Ala 180 185 190 gca aga gcaggt gtt tac aac ctc acc aaa tct tta gct ttg gaa tgg 624 Ala Arg Ala GlyVal Tyr Asn Leu Thr Lys Ser Leu Ala Leu Glu Trp 195 200 205 gcc tgc agtgga ata cgg atc aat tgt gtt gcc cct gga gtt att tat 672 Ala Cys Ser GlyIle Arg Ile Asn Cys Val Ala Pro Gly Val Ile Tyr 210 215 220 tcc cag actgct gtg gag aac tat ggt tcc tgg gga caa agc ttc ttt 720 Ser Gln Thr AlaVal Glu Asn Tyr Gly Ser Trp Gly Gln Ser Phe Phe 225 230 235 240 gaa gggtct ttt cag aaa atc ccc gct aaa cga att ggt gtt cct gag 768 Glu Gly SerPhe Gln Lys Ile Pro Ala Lys Arg Ile Gly Val Pro Glu 245 250 255 gag gtctcc tct gtg gtc tgc ttc cta ctg tct cct gca gct tcc ttc 816 Glu Val SerSer Val Val Cys Phe Leu Leu Ser Pro Ala Ala Ser Phe 260 265 270 atc actgga cag tcg gtg gat gtg gat ggg ggc cgg agt ctc tat act 864 Ile Thr GlyGln Ser Val Asp Val Asp Gly Gly Arg Ser Leu Tyr Thr 275 280 285 cac tcgtat gag gta cca gat cat gac aac tgg ccc aag gga gca ggg 912 His Ser TyrGlu Val Pro Asp His Asp Asn Trp Pro Lys Gly Ala Gly 290 295 300 gac ctttct gtt gtc aaa aag atg aag gag acc tta aag gag aaa gct 960 Asp Leu SerVal Val Lys Lys Met Lys Glu Thr Leu Lys Glu Lys Ala 305 310 315 320 aagctc 966 Lys Leu 13 303 PRT Rattus norvegicus 13 Met Gly Ser Trp Lys SerGly Gln Ser Tyr Leu Ala Ala Gly Leu Leu 1 5 10 15 Gln Asn Gln Val AlaVal Val Thr Gly Gly Ala Thr Gly Ile Gly Lys 20 25 30 Ala Ile Ser Arg GluLeu Leu His Leu Gly Cys Asn Val Val Ile Ala 35 40 45 Ser Arg Lys Leu AspArg Leu Thr Ala Ala Val Asp Glu Leu Arg Ala 50 55 60 Ser Gln Pro Pro SerSer Ser Thr Gln Val Thr Ala Ile Gln Cys Asn 65 70 75 80 Ile Arg Lys GluGlu Glu Val Asn Asn Leu Val Lys Ser Thr Leu Ala 85 90 95 Lys Tyr Gly LysIle Asn Phe Leu Val Asn Asn Ala Gly Gly Gln Phe 100 105 110 Met Ala ProAla Glu Asp Ile Thr Ala Lys Gly Trp Gln Ala Val Ile 115 120 125 Glu ThrAsn Leu Thr Gly Thr Phe Tyr Met Cys Lys Ala Val Tyr Asn 130 135 140 SerTrp Met Lys Asp His Gly Gly Ser Ile Val Asn Ile Ile Val Leu 145 150 155160 Leu Asn Asn Gly Phe Pro Thr Ala Ala His Ser Gly Ala Ala Arg Ala 165170 175 Gly Val Tyr Asn Leu Thr Lys Thr Met Ala Leu Thr Trp Ala Ser Ser180 185 190 Gly Val Arg Ile Asn Cys Val Ala Pro Gly Thr Ile Tyr Ser GlnThr 195 200 205 Ala Val Asp Asn Tyr Gly Glu Leu Gly Gln Thr Met Phe GluMet Ala 210 215 220 Phe Glu Asn Ile Pro Ala Lys Arg Val Gly Leu Pro GluGlu Ile Ser 225 230 235 240 Pro Leu Val Cys Phe Leu Leu Ser Pro Ala AlaSer Phe Ile Thr Gly 245 250 255 Gln Leu Ile Asn Val Asp Gly Gly Gln AlaLeu Tyr Thr Arg Asn Phe 260 265 270 Thr Ile Pro Asp His Asp Asn Trp ProVal Gly Ala Gly Asp Ser Ser 275 280 285 Phe Ile Lys Lys Val Lys Glu SerLeu Lys Lys Gln Ala Arg Leu 290 295 300 14 1108 DNA Mus musculus CDS(102)..(1034) 14 ggaatggatg ctgttggctt aaacctcccc ctgccctggg ggttgcaaccagggtctctg 60 caaagccaat cctttgtcat cccgctgtcc tgcagagcaa g atg ggg ctcatg gct 116 Met Gly Leu Met Ala 1 5 gtc ctg atg cta ccc ctg ctg ctg ctggga atc agc ggc ctc ctc ttc 164 Val Leu Met Leu Pro Leu Leu Leu Leu GlyIle Ser Gly Leu Leu Phe 10 15 20 att tac cag gag gca tcc agg ctg tgg tcgaag tct gcc gtg cag aac 212 Ile Tyr Gln Glu Ala Ser Arg Leu Trp Ser LysSer Ala Val Gln Asn 25 30 35 aaa gtg gtg gtc atc aca gat gcc atc tca ggactg gga aag gag tgt 260 Lys Val Val Val Ile Thr Asp Ala Ile Ser Gly LeuGly Lys Glu Cys 40 45 50 gct cgg gtg ttc cat gca ggt ggg gca agg ctg gtgctg tgt gga aag 308 Ala Arg Val Phe His Ala Gly Gly Ala Arg Leu Val LeuCys Gly Lys 55 60 65 aac tgg gag gga ctg gag agc ctc tat gcc acc ttg accagt gtg gct 356 Asn Trp Glu Gly Leu Glu Ser Leu Tyr Ala Thr Leu Thr SerVal Ala 70 75 80 85 gac ccc agc aag aca ttc acc ccc aag ctg gtc ctc ctggat ctc tca 404 Asp Pro Ser Lys Thr Phe Thr Pro Lys Leu Val Leu Leu AspLeu Ser 90 95 100 gac att agc tgt gtt caa gat gtg gcc aaa gag gtc ctggac tgc tac 452 Asp Ile Ser Cys Val Gln Asp Val Ala Lys Glu Val Leu AspCys Tyr 105 110 115 ggc tgt gtg gac atc ctc atc aac aat gcc agc gtg aaagtg aag ggg 500 Gly Cys Val Asp Ile Leu Ile Asn Asn Ala Ser Val Lys ValLys Gly 120 125 130 cct gcc cac aag att tcc ctg gag ctt gac aaa aag atcatg gat gcc 548 Pro Ala His Lys Ile Ser Leu Glu Leu Asp Lys Lys Ile MetAsp Ala 135 140 145 aac tac ttc gga ccc atc act tta acc aaa gtt ctg cttccc aac atg 596 Asn Tyr Phe Gly Pro Ile Thr Leu Thr Lys Val Leu Leu ProAsn Met 150 155 160 165 atc tcc agg aga aca ggc cag att gtg tta gtg aacaac atc caa gcg 644 Ile Ser Arg Arg Thr Gly Gln Ile Val Leu Val Asn AsnIle Gln Ala 170 175 180 aag ttt gga atc ccg ttc cgc aca gct tat gca gcctct aag cat gcc 692 Lys Phe Gly Ile Pro Phe Arg Thr Ala Tyr Ala Ala SerLys His Ala 185 190 195 gtc atg ggc ttc ttt gac tgc ctc cga gcc gag gttgag gaa tac gat 740 Val Met Gly Phe Phe Asp Cys Leu Arg Ala Glu Val GluGlu Tyr Asp 200 205 210 gtt gtg gtc agc acc gtg agc cca act ttc atc cgctcc tac cgt gct 788 Val Val Val Ser Thr Val Ser Pro Thr Phe Ile Arg SerTyr Arg Ala 215 220 225 tcc cct gag caa aga aac tgg gag aca tcc att tgtaaa ttc ttc tgc 836 Ser Pro Glu Gln Arg Asn Trp Glu Thr Ser Ile Cys LysPhe Phe Cys 230 235 240 245 agg aag cta gcc tat ggc gtg cac ccg gtg gaggtg gct gag gaa gtg 884 Arg Lys Leu Ala Tyr Gly Val His Pro Val Glu ValAla Glu Glu Val 250 255 260 atg cgc aca gta cgg agg aag aag caa gag gtgttc atg gcc aac ccg 932 Met Arg Thr Val Arg Arg Lys Lys Gln Glu Val PheMet Ala Asn Pro 265 270 275 gtt cct aag gct gcc gtg ttc atc cgc acc ttcttc cct gag ttc ttc 980 Val Pro Lys Ala Ala Val Phe Ile Arg Thr Phe PhePro Glu Phe Phe 280 285 290 ttc gct gtg gtg gcc tgt ggg gtg aag gag aagctc aat gtc cca gaa 1028 Phe Ala Val Val Ala Cys Gly Val Lys Glu Lys LeuAsn Val Pro Glu 295 300 305 gag ggt taacctcgtg gccaaagggg tcactcaaggggaataaagg ctttcctaga 1084 Glu Gly 310 gaaaaaaaaa aaaaaaaaaa aaaa 110815 311 PRT Mus musculus 15 Met Gly Leu Met Ala Val Leu Met Leu Pro LeuLeu Leu Leu Gly Ile 1 5 10 15 Ser Gly Leu Leu Phe Ile Tyr Gln Glu AlaSer Arg Leu Trp Ser Lys 20 25 30 Ser Ala Val Gln Asn Lys Val Val Val IleThr Asp Ala Ile Ser Gly 35 40 45 Leu Gly Lys Glu Cys Ala Arg Val Phe HisAla Gly Gly Ala Arg Leu 50 55 60 Val Leu Cys Gly Lys Asn Trp Glu Gly LeuGlu Ser Leu Tyr Ala Thr 65 70 75 80 Leu Thr Ser Val Ala Asp Pro Ser LysThr Phe Thr Pro Lys Leu Val 85 90 95 Leu Leu Asp Leu Ser Asp Ile Ser CysVal Gln Asp Val Ala Lys Glu 100 105 110 Val Leu Asp Cys Tyr Gly Cys ValAsp Ile Leu Ile Asn Asn Ala Ser 115 120 125 Val Lys Val Lys Gly Pro AlaHis Lys Ile Ser Leu Glu Leu Asp Lys 130 135 140 Lys Ile Met Asp Ala AsnTyr Phe Gly Pro Ile Thr Leu Thr Lys Val 145 150 155 160 Leu Leu Pro AsnMet Ile Ser Arg Arg Thr Gly Gln Ile Val Leu Val 165 170 175 Asn Asn IleGln Ala Lys Phe Gly Ile Pro Phe Arg Thr Ala Tyr Ala 180 185 190 Ala SerLys His Ala Val Met Gly Phe Phe Asp Cys Leu Arg Ala Glu 195 200 205 ValGlu Glu Tyr Asp Val Val Val Ser Thr Val Ser Pro Thr Phe Ile 210 215 220Arg Ser Tyr Arg Ala Ser Pro Glu Gln Arg Asn Trp Glu Thr Ser Ile 225 230235 240 Cys Lys Phe Phe Cys Arg Lys Leu Ala Tyr Gly Val His Pro Val Glu245 250 255 Val Ala Glu Glu Val Met Arg Thr Val Arg Arg Lys Lys Gln GluVal 260 265 270 Phe Met Ala Asn Pro Val Pro Lys Ala Ala Val Phe Ile ArgThr Phe 275 280 285 Phe Pro Glu Phe Phe Phe Ala Val Val Ala Cys Gly ValLys Glu Lys 290 295 300 Leu Asn Val Pro Glu Glu Gly 305 310 16 933 DNAMus musculus CDS (1)..(933) 16 atg ggg ctc atg gct gtc ctg atg cta cccctg ctg ctg ctg gga atc 48 Met Gly Leu Met Ala Val Leu Met Leu Pro LeuLeu Leu Leu Gly Ile 1 5 10 15 agc ggc ctc ctc ttc att tac cag gag gcatcc agg ctg tgg tcg aag 96 Ser Gly Leu Leu Phe Ile Tyr Gln Glu Ala SerArg Leu Trp Ser Lys 20 25 30 tct gcc gtg cag aac aaa gtg gtg gtc atc acagat gcc atc tca gga 144 Ser Ala Val Gln Asn Lys Val Val Val Ile Thr AspAla Ile Ser Gly 35 40 45 ctg gga aag gag tgt gct cgg gtg ttc cat gca ggtggg gca agg ctg 192 Leu Gly Lys Glu Cys Ala Arg Val Phe His Ala Gly GlyAla Arg Leu 50 55 60 gtg ctg tgt gga aag aac tgg gag gga ctg gag agc ctctat gcc acc 240 Val Leu Cys Gly Lys Asn Trp Glu Gly Leu Glu Ser Leu TyrAla Thr 65 70 75 80 ttg acc agt gtg gct gac ccc agc aag aca ttc acc cccaag ctg gtc 288 Leu Thr Ser Val Ala Asp Pro Ser Lys Thr Phe Thr Pro LysLeu Val 85 90 95 ctc ctg gat ctc tca gac att agc tgt gtt caa gat gtg gccaaa gag 336 Leu Leu Asp Leu Ser Asp Ile Ser Cys Val Gln Asp Val Ala LysGlu 100 105 110 gtc ctg gac tgc tac ggc tgt gtg gac atc ctc atc aac aatgcc agc 384 Val Leu Asp Cys Tyr Gly Cys Val Asp Ile Leu Ile Asn Asn AlaSer 115 120 125 gtg aaa gtg aag ggg cct gcc cac aag att tcc ctg gag cttgac aaa 432 Val Lys Val Lys Gly Pro Ala His Lys Ile Ser Leu Glu Leu AspLys 130 135 140 aag atc atg gat gcc aac tac ttc gga ccc atc act tta accaaa gtt 480 Lys Ile Met Asp Ala Asn Tyr Phe Gly Pro Ile Thr Leu Thr LysVal 145 150 155 160 ctg ctt ccc aac atg atc tcc agg aga aca ggc cag attgtg tta gtg 528 Leu Leu Pro Asn Met Ile Ser Arg Arg Thr Gly Gln Ile ValLeu Val 165 170 175 aac aac atc caa gcg aag ttt gga atc ccg ttc cgc acagct tat gca 576 Asn Asn Ile Gln Ala Lys Phe Gly Ile Pro Phe Arg Thr AlaTyr Ala 180 185 190 gcc tct aag cat gcc gtc atg ggc ttc ttt gac tgc ctccga gcc gag 624 Ala Ser Lys His Ala Val Met Gly Phe Phe Asp Cys Leu ArgAla Glu 195 200 205 gtt gag gaa tac gat gtt gtg gtc agc acc gtg agc ccaact ttc atc 672 Val Glu Glu Tyr Asp Val Val Val Ser Thr Val Ser Pro ThrPhe Ile 210 215 220 cgc tcc tac cgt gct tcc cct gag caa aga aac tgg gagaca tcc att 720 Arg Ser Tyr Arg Ala Ser Pro Glu Gln Arg Asn Trp Glu ThrSer Ile 225 230 235 240 tgt aaa ttc ttc tgc agg aag cta gcc tat ggc gtgcac ccg gtg gag 768 Cys Lys Phe Phe Cys Arg Lys Leu Ala Tyr Gly Val HisPro Val Glu 245 250 255 gtg gct gag gaa gtg atg cgc aca gta cgg agg aagaag caa gag gtg 816 Val Ala Glu Glu Val Met Arg Thr Val Arg Arg Lys LysGln Glu Val 260 265 270 ttc atg gcc aac ccg gtt cct aag gct gcc gtg ttcatc cgc acc ttc 864 Phe Met Ala Asn Pro Val Pro Lys Ala Ala Val Phe IleArg Thr Phe 275 280 285 ttc cct gag ttc ttc ttc gct gtg gtg gcc tgt ggggtg aag gag aag 912 Phe Pro Glu Phe Phe Phe Ala Val Val Ala Cys Gly ValLys Glu Lys 290 295 300 ctc aat gtc cca gaa gag ggt 933 Leu Asn Val ProGlu Glu Gly 305 310

What is claimed:
 1. An isolated nucleic acid molecule selected from thegroup consisting of: (a) a nucleic acid molecule comprising thenucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7,SEQ ID NO:10, or SEQ ID NO:14; and (b) a nucleic acid moleculecomprising the nucleotide sequence set forth in SEQ ID NO:3, SEQ IDNO:6, SEQ ID NO:9, SEQ ID NO:12, or SEQ ID NO:16.
 2. An isolated nucleicacid molecule which encodes a polypeptide comprising the amino acidsequence set forth in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ IDNO:11, or SEQ ID NO:15.
 3. An isolated nucleic acid molecule comprisingthe nucleotide sequence contained in the plasmid deposited with ATCC® asAccession Number ______, PTA-1845, ______, ______, or ______.
 4. Anisolated nucleic acid molecule which encodes a naturally occurringallelic variant of a polypeptide comprising the amino acid sequence setforth in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, or SEQ IDNO:15.
 5. An isolated nucleic acid molecule selected from the groupconsisting of: a) a nucleic acid molecule comprising a nucleotidesequence which is at least 60% identical to the nucleotide sequence ofSEQ ID NO:1 or 3, SEQ ID NO:4 or 6, SEQ ID NO:7 or 9, SEQ ID NO:10 or12, or SEQ ID NO:14 or 16, or a complement thereof; b) a nucleic acidmolecule comprising a fragment of at least 50 nucleotides of a nucleicacid comprising the nucleotide sequence of SEQ ID NO:1 or 3, SEQ ID NO:4or 6, SEQ ID NO:7 or 9, SEQ ID NO:10 or 12, or SEQ ID NO:14 or 16, or acomplement thereof; c) a nucleic acid molecule which encodes apolypeptide comprising an amino acid sequence at least about 60%identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:8, SEQ ID NO:11, or SEQ ID NO:15; and d) a nucleic acid moleculewhich encodes a fragment of a polypeptide comprising the amino acidsequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, or SEQID NO:15, wherein the fragment comprises at least 16 contiguous aminoacid residues of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5,SEQ ID NO:8, SEQ ID NO:11, or SEQ ID NO:15.
 6. An isolated nucleic acidmolecule which hybridizes to the nucleic acid molecule of any one ofclaims 1, 2, 3, 4, or 5 under stringent conditions.
 7. An isolatednucleic acid molecule comprising a nucleotide sequence which iscomplementary to the nucleotide sequence of the nucleic acid molecule ofany one of claims 1, 2, 3, 4, or
 5. 8. An isolated nucleic acid moleculecomprising the nucleic acid molecule of any one of claims 1, 2, 3, 4, or5, and a nucleotide sequence encoding a heterologous polypeptide.
 9. Avector comprising the nucleic acid molecule of any one of claims 1, 2,3, 4, or
 5. 10. The vector of claim 9, which is an expression vector.11. A host cell transfected with the expression vector of claim
 10. 12.A method of producing a polypeptide comprising culturing the host cellof claim 11 in an appropriate culture medium to, thereby, produce thepolypeptide.
 13. An isolated polypeptide selected from the groupconsisting of: a) a fragment of a polypeptide comprising the amino acidsequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, or SEQID NO:15, wherein the fragment comprises at least 16 contiguous aminoacids of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, or SEQ IDNO:15; b) a naturally occurring allelic variant of a polypeptidecomprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ IDNO:8, SEQ ID NO:11, or SEQ ID NO:15, wherein the polypeptide is encodedby a nucleic acid molecule which hybridizes to a complement of a nucleicacid molecule consisting of SEQ ID NO:1 or 3, SEQ ID NO:4 or 6, SEQ IDNO:7 or 9, SEQ ID NO:10 or 12, or SEQ ID NO:14 or 16 under stringentconditions; c) a polypeptide which is encoded by a nucleic acid moleculecomprising a nucleotide sequence which is at least 60% identical to anucleic acid comprising the nucleotide sequence of SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16; d) a polypeptide comprisingan amino acid sequence which is at least 60% identical to the amino acidsequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, or SEQID NO:15.
 14. The isolated polypeptide of claim 13 comprising the aminoacid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:11.15. The polypeptide of claim 13, further comprising heterologous aminoacid sequences.
 16. An antibody which selectively binds to a polypeptideof claim
 13. 17. A method for detecting the presence of a polypeptide ofclaim 13 in a sample comprising: a) contacting the sample with acompound which selectively binds to the polypeptide; and b) determiningwhether the compound binds to the polypeptide in the sample to therebydetect the presence of a polypeptide of claim 13 in the sample.
 18. Themethod of claim 17, wherein the compound which binds to the polypeptideis an antibody.
 19. A kit comprising a compound which selectively bindsto a polypeptide of claim 13 and instructions for use.
 20. A method fordetecting the presence of a nucleic acid molecule of any one of claims1, 2, 3, 4, or 5 in a sample comprising: a) contacting the sample with anucleic acid probe or primer which selectively hybridizes to the nucleicacid molecule; and b) determining whether the nucleic acid probe orprimer binds to a nucleic acid molecule in the sample to thereby detectthe presence of a nucleic acid molecule of any one of claims 1, 2, 3, 4,or 5 in the sample.
 21. The method of claim 20, wherein the samplecomprises mRNA molecules and is contacted with a nucleic acid probe. 22.A kit comprising a compound which selectively hybridizes to a nucleicacid molecule of any one of claims 1, 2, 3, 4, or 5 and instructions foruse.
 23. A method for identifying a compound which binds to apolypeptide of claim 13 comprising: a) contacting the polypeptide, or acell expressing the polypeptide with a test compound; and b) determiningwhether the polypeptide binds to the test compound.
 24. The method ofclaim 23, wherein the binding of the test compound to the polypeptide isdetected by a method selected from the group consisting of: a) detectionof binding by direct detection of test compound/polypeptide binding; b)detection of binding using a competition binding assay; and c) detectionof binding using an assay for DHDR activity.
 25. A method for modulatingthe activity of a polypeptide of claim 13 comprising contacting thepolypeptide or a cell expressing the polypeptide with a compound whichbinds to the polypeptide in a sufficient concentration to modulate theactivity of the polypeptide.
 26. A method for identifying a compoundwhich modulates the activity of a polypeptide of claim 13 comprising: a)contacting a polypeptide of claim 13 with a test compound; and b)determining the effect of the test compound on the activity of thepolypeptide to thereby identify a compound which modulates the activityof the polypeptide.
 27. The method of claim 26, wherein said activity ismodulation of virus activity.
 28. A method for identifying a compoundwhich modulates virus activity comprising: a) contacting the polypeptideof claim 13, or a cell expressing the polypeptide with a test compound;and b) identifying the compound as a modulator of virus activity bydetermining the effect of the test compound on the activity of thepolypeptide.
 29. A method for treating a subject having a viral disordercomprising administering to the subject a DHDR modulator, therebytreating said subject having a viral disorder.
 30. A method for treatinga subject having viral disorder comprising administering to the subjecta DHDR modulator, wherein the DHDR modulator is the modulator identifiedby the method of claim 26, thereby treating said subject having a viraldisorder.
 31. The method of claim 29, wherein the DHDR modulator is asmall molecule.
 32. The method of claim 29, wherein the DHDR modulatoris administered in a pharmaceutically acceptable formulation.
 33. Themethod of claim 29, wherein the DHDR modulator is administered using agene therapy vector.
 34. The method of claim 29, wherein the DHDRmodulator is capable of modulating DHDR polypeptide activity.
 35. Themethod of claim 26, wherein said activity is modulation of cellularproliferation.
 36. A method for identifying a compound which modulatescellular proliferation comprising: a) contacting the polypeptide ofclaim 13, or a cell expressing the polypeptide with a test compound; andb) identifying the compound as a modulator of cellular proliferation bydetermining the effect of the test compound on the activity of thepolypeptide.
 37. A method for treating a subject having a cellularproliferation disorder comprising administering to the subject a DHDRmodulator, thereby treating said subject having a cellular proliferationdisorder.
 38. A method for treating a subject having cellularproliferation disorder comprising administering to the subject a DHDRmodulator, wherein the DHDR modulator is the modulator identified by themethod of claim 26, thereby treating said subject having a cellularproliferation disorder.
 39. The method of claim 37, wherein the DHDRmodulator is a small molecule.
 40. The method of claim 37, wherein theDHDR modulator is administered in a pharmaceutically acceptableformulation.
 41. The method of claim 37, wherein the DHDR modulator isadministered using a gene therapy vector.
 42. The method of 37, whereinthe DHDR modulator is capable of modulating DHDR polypeptide activity.